Background
Oropharyngeal squamous cell carcinomas (OpSCCs) are commonly associated with high rates of treatment failure.
Objectives
To evaluate methylation‐based markers in plasma from OpSCC patients ...as emerging tools for accurate/noninvasive follow‐up.
Methods
Pretreatment formalin‐fixed paraffin‐embedded (FFPE) biopsies (n = 52) and paired plasma (n = 15) were tested for the methylation of CCNA1, DAPK, CDH8, and TIMP3 by droplet digital PCR (ddPCR).
Results
Seventy‐one percent (37/52) of the biopsies showed methylation of at least one of the evaluated genes and tumor CCNA1 methylation was associated with recurrence‐free survival. Methylated circulating tumor DNA (meth‐ctDNA) was detected in 11/15 (73.3%) plasma samples; conversely, plasma samples from healthy controls were all negative for DNA methylation (area under the curve = 0.867; 95% confidence interval = 0.720‐1.000). Additionally, preliminary results on the detection of meth‐ctDNA in plasma collected during follow‐up closely matched patient outcome.
Conclusions
The results suggest the feasibility of detecting meth‐ctDNA in plasma using ddPCR and a possible application on routine setting after further validation.
Medulloblastoma is the most frequent malignant brain tumor in children. Four medulloblastoma molecular subgroups, MBSHH, MBWNT, MBGRP3 and MBGRP4, have been identified by integrated high‐throughput ...platforms. Recently, a 22‐gene panel NanoString‐based assay was developed for medulloblastoma molecular subgrouping, but the robustness of this assay has not been widely evaluated. Mutations in the gene for human telomerase reverse transcriptase (hTERT) have been found in medulloblastomas and are associated with distinct molecular subtypes. This study aimed to implement the 22‐gene panel in a Brazilian context, and to associate the molecular profile with patients’ clinical‐pathological features. Formalin‐fixed, paraffin‐embedded (FFPE) medulloblastoma samples (n = 104) from three Brazilian centers were evaluated. Expression profiling of the 22‐gene panel was performed by NanoString and a Canadian series (n = 240) was applied for training phase. hTERT mutations were analyzed by PCR followed by direct Sanger sequencing and the molecular profile was associated with patients’ clinicopathological features. Overall, 65% of the patients were male, average age at diagnosis was 18 years and 7% of the patients presented metastasis at diagnosis. The molecular classification was attained in 100% of the cases, with the following frequencies: MBSHH (n = 51), MBWNT (n = 19), MBGRP4 (n = 19) and MBGRP3 (n = 15). The MBSHH and MBGRP3 subgroups were associated with older and younger patients, respectively. The MBGRP4 subgroup exhibited the lowest 5‐year cancer‐specific overall survival (OS), yet in the multivariate analysis, only metastasis at diagnosis and surgical resection were associated with OS. hTERT mutations were detected in 29% of the cases and were associated with older patients, increased hTERT expression and MBSHH subgroup. The 22‐gene panel provides a reproducible assay for molecular subgrouping of medulloblastoma FFPE samples in a routine setting and is well‐suited for future clinical trials.
Human hotspot TERT promoter (TERTp) mutations have been reported in a wide range of tumours. Several studies have shown that TERTp mutations are associated with clinicopathological features; in some ...instances, TERTp mutations were considered as biomarkers of poor prognosis. The rs2853669 SNP, located in the TERT promoter region, was reported to modulate the increased TERT expression levels induced by the recurrent somatic mutations. In this study we aimed to determine the frequency and prognostic value of TERTp mutations and TERT rs2853669 SNP in 504 gliomas from Portuguese and Brazilian patients. TERTp mutations were detected in 47.8% of gliomas (216/452). Glioblastomas (GBM) exhibited the highest frequency of TERTp mutations (66.9%); in this glioma subtype, we found a significant association between TERTp mutations and poor prognosis, regardless of the population. Moreover, in a multivariate analysis, TERTp mutations were the only independent prognostic factor. Our data also showed that the poor prognosis conferred by TERTp mutations was restricted to GBM patients carrying the rs2853669 A allele and not in those carrying the G allele. In conclusion, the presence of TERTp mutations was associated with worse prognosis in GBM patients, although such association depended on the status of the rs2853669 SNP. The status of the rs2853669 SNP should be taken in consideration when assessing the prognostic value of TERTp mutations in GBM patients. TERTp mutations and the rs2853669 SNP can be used in the future as biomarkers of glioma prognosis.
What's new?
Cancer cells avoid senescence in part by reactivating telomerase (TERT), a ribonucleoprotein that replenishes shortening telomeres. Here, the authors discover a positive association between TERT promoter mutations and unfavorable prognosis in glioblastoma patients from Portuguese and Brazilian origin. This association was only observed in patients with a specific allelic background (AA) in a TERT polymorphism (rs2853669) recently linked to enhanced TERT mRNA levels. The authors recommend considering the allelic status of rs2853669 when assessing the prognostic value of TERT promoter mutations in glioblastoma patients.
Tobacco- or human papillomavirus- driven oropharyngeal squamous cell carcinomas (OpSCC) represent distinct clinical, biological and epidemiological entities. The aim of this study was to identify ...genetic variants based on somatic alterations in OpSCC samples from an admixed population, and to test for association with clinical features. The entire coding region of 15 OpSCC driver genes was sequenced by next-generation sequencing in 51 OpSCC FFPE samples. Thirty-five percent of the patients (18/51) were HPV-positive and current or past tobacco consumption was reported in 86.3% (44/51). The mutation profile identified an average of 2.67 variants per sample. Sixty-three percent of patients (32/51; 62.7%) were mutated for at least one of the genes tested and TP53 was the most frequently mutated gene. The presence of mutation in NOTCH1 and PTEN, significantly decreased patient's recurrence-free survival, but only NOTCH1 mutation remained significant after stepwise selection, with a risk of recurrence of 4.5 (HR 95% CI = 1.11-14.57; Cox Regression p = 0.034). These results show that Brazilian OpSCC patients exhibit a similar clinical and genetic profile in comparison to other populations. Molecular characterization is a promising tool for the definition of clinical subgroups, aiding in a more precise tailoring of treatment and prognostication.
Rosette-forming glioneuronal tumor (RGNT) of the IV ventricle is a rare and recently recognized brain tumor entity. It is histologically composed by two distinct features: a glial component, ...resembling pilocytic astrocytoma, and a component forming neurocytic rosettes and/or perivascular rosettes. Herein, we describe a 33-year-old man with RGNT arising in the spinal cord. Following an immunohistochemistry validation, we further performed an extensive genomic analysis, using array-CGH (aCGH), whole exome and cancer-related hotspot sequencing, in order to better understand its underlying biology. We observed the loss of 1p and gain of 1q, as well as gain of the whole chromosomes 7, 9 and 16. Local amplifications in 9q34.2 and 19p13.3 (encompassing the gene SBNO2) were identified. Moreover, we observed focal gains/losses in several chromosomes. Additionally, on chromosome 7, we identified the presence of the KIAA1549:BRAF gene fusion, which was further validated by RT-PCR and FISH. Across all mutational analyses, we detected and validated the somatic mutations of the genes MLL2, CNNM3, PCDHGC4 and SCN1A. Our comprehensive molecular profiling of this RGNT suggests that MAPK pathway and methylome changes, driven by KIAA1549:BRAF fusion and MLL2 mutation, respectively, could be associated with the development of this rare tumor entity.
The identification of molecular markers in negative surgical margins of oral squamous cell carcinoma (OSCC) might help in identifying residual molecular aberrations, and potentially improve the ...prediction of prognosis. We performed an Infinium MethylationEPIC BeadChip array on 32 negative surgical margins stratified based on the status of tumor recurrence in order to identify recurrence-specific aberrant DNA methylation (DNAme) markers. We identified 2512 recurrence-associated Differentially Methylated Positions (DMPs) and 392 Differentially Methylated Regions (DMRs) which were enriched in cell signaling and cancer-related pathways. A set of 14-CpG markers was able to discriminate recurrent and non-recurrent cases with high specificity and sensitivity rates (AUC 0.98,
= 3 × 10
; CI: 0.95-1). A risk score based on the 14-CpG marker panel was applied, with cases classified within higher risk scores exhibiting poorer survival. The results were replicated using tumor-adjacent normal HNSCC samples from The Cancer Genome Atlas (TCGA). We identified residual DNAme aberrations in the negative surgical margins of OSCC patients, which could be informative for patient management by improving therapeutic intervention. This study proposes a novel DNAme-based 14-CpG marker panel as a promising predictor for tumor recurrence, which might contribute to improved decision-making for the personalized treatment of OSCC cases.
SPINT2 Deregulation in Prostate Carcinoma Pereira, Márcia Santos; de Almeida, Gisele Caravina; Pinto, Filipe ...
The journal of histochemistry and cytochemistry,
01/2016, Volume:
64, Issue:
1
Journal Article
Peer reviewed
Open access
SPINT2 is a tumor suppressor gene that inhibits proteases implicated in cancer progression, like HGFA, hepsin and matriptase. Loss of SPINT2 expression in tumors has been associated with gene ...promoter hypermethylation; however, little is known about the mechanisms of SPINT2 deregulation in prostate cancer (PCa). We aimed to analyze SPINT2 expression levels and understand the possible regulation by SPINT2 promoter hypermethylation in PCa. In a cohort of 57 cases including non-neoplastic and PCa tissues, SPINT2 expression and promoter methylation was analyzed by immunohistochemistry and methylation-specific PCR, respectively. Methylation status of the SPINT2 promoter was also evaluated by bisulfite sequencing and 5-aza-2’-deoxycytidine treatment. Oncomine and TCGA databases were used to perform in silico PCa analysis of SPINT2 mRNA and methylation levels. A reduction in SPINT2 expression levels from non-neoplastic to PCa tissues was observed; however, none of the cases exhibited SPINT2 promoter methylation. Both bisulfite sequencing and 5-aza demonstrated that SPINT2 promoter is not methylated in PCa cells. Bioinformatics approaches did not show downregulation of SPINT2 at the mRNA level and, in corroboration with our results, SPINT2 promoter region is reported to be unmethylated. Our study suggests an involvement of SPINT2 in PCa tumorigenesis, probably in association with a post-translational regulation of SPINT2.
Purpose
High-grade gliomas (HGG) remain one of the most aggressive tumors, which is primarily due to its diffuse infiltrative nature. Serine proteases and metalloproteases are known to play key roles ...in cellular migration and invasion mechanisms. SPINT2, also known as HAI-2, is an important serine protease inhibitor that can affect MET signaling. SPINT2 has been found to be frequently downregulated in various tumors, whereby hypermethylation of its promoter appears to serve as a common mechanism. Here, we assessed the clinical relevance of SPINT2 expression and promoter hypermethylation in pediatric and adult HGG and explored its functional role.
Methods
A series of 371 adult and 77 pediatric primary HGG samples was assessed for SPINT2 protein expression (immunohistochemistry) and promoter methylation (methylation-specific PCR) patterns. After
SPINT2
knockdown and knock-in in adult and pediatric HGG cell lines, a variety of
in vitro
assays was carried out to determine the role of SPINT2 in glioma cell viability and invasion, as well as their mechanistic associations with metalloprotease activities.
Results
We found that SPINT2 protein expression was frequently absent in adult (85.3%) and pediatric (100%) HGG samples. The
SPINT2
gene promoter was found to be hypermethylated in approximately half of both adult and pediatric gliomas. Through functional assays we revealed a suppressor activity of SPINT2 in glioma cell proliferation and viability, as well as in their migration and invasion. These functions appear to be mediated in part by MMP2 expression and activity.
Conclusions
We conclude that dysregulation of SPINT2 is a common event in both pediatric and adult HGG, in which SPINT2 may act as a tumor suppressor.
Tobacco or human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas (OPSCC) represent different clinical and epidemiologic entities. This study investigated the prevalence of ...HPV-positive and HPV-negative OPSCC in a reference cancer hospital in Brazil and its association with clinical and demographic data, as well as its impact on overall survival.
HPV infection was determined by p16-IHC in pre-treatment formalin-fixed paraffin-embedded samples from all patients with OPSCC diagnosed at Barretos Cancer Hospital between 2008 and 2018. The prevalence of HPV-positive cases and its temporal trend was assessed, and the association of clinical and demographic data with HPV infection and the impact on patient overall survival was evaluated.
A total of 797 patients with OPSCC were included in the study. The prevalence of HPV-associated tumors in the period was 20.6% 95% confidence interval, 17.5-24.0 with a significant trend for increase of HPV-positive cases over the years (annual percentage change = 12.87). In a multivariate analysis, the variables gender, level of education, smoking, tumor sublocation, region of Brazil, and tumor staging had a significant impact in HPV positivity, and a greater overall survival (OS) was observed in HPV-positive patients (5-year OS: 47.9% vs. 22.0%;
= 0.0001).
This study represents the largest cohort of Brazilian patients with OPSCC characterized according to HPV status. We report significant differences in demographics and clinical presentation according to HPV status, and an increasing trend in prevalence for HPV-induced tumors.
These findings can potentially contribute to a better stratification and management of patients as well as assist in prevention strategies.
The 5'-methylthioadenosine phosphorylase (MTAP) gene is located in the chromosomal region 9p21.
deletion is a frequent event in a wide variety of human cancers; however, its biological role in ...tumorigenesis remains unclear. The purpose of this study was to characterize the MTAP expression profile in a series of gliomas and to associate it with patients' clinicopathological features. Moreover, we sought to evaluate, through glioma gene-edited cell lines, the biological impact of MTAP in gliomas. MTAP expression was evaluated in 507 glioma patients by immunohistochemistry (IHC), and the expression levels were associated with patients' clinicopathological features. Furthermore, an in silico study was undertaken using genomic databases totalizing 350 samples. In glioma cell lines, MTAP was edited, and following MTAP overexpression and knockout (KO), a transcriptome analysis was performed by NanoString Pan-Cancer Pathways panel. Moreover, MTAP's role in glioma cell proliferation, migration, and invasion was evaluated. Homozygous deletion of 9p21 locus was associated with a reduction of
mRNA expression in the TCGA (The Cancer Genome Atlas) - glioblastoma dataset (p < 0.01). In addition, the loss of
expression was markedly high in high-grade gliomas (46.6% of cases) determined by IHC and Western blotting (40% of evaluated cell lines). Reduced
expression was associated with a better prognostic in the adult glioblastoma dataset (
< 0.001). Nine genes associated with five pathways were differentially expressed in MTAP-knockout (KO) cells, with six upregulated and three downregulated in MTAP. Analysis of cell proliferation, migration, and invasion did not show any significant differences between MTAP gene-edited and control cells. Our results integrating data from patients as well as in silico and in vitro models provide evidence towards the lack of strong biological importance of MTAP in gliomas. Despite the frequent loss of MTAP, it seems not to have a clinical impact in survival and does not act as a canonic tumor suppressor gene in gliomas.
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