Relevance of mutations in protein deubiquitinases genes and TP53 in corticotroph pituitary tumors.

Introduction: Corticotroph pituitary neuroendocrine tumors (PitNETs) develop from ACTH-producing cells. They commonly cause Cushing's disease (CD), however, some remain clinically silent. Recurrent USP8, USP48, BRAF and TP53 mutations occur in corticotroph PitNETs. The aim of our study was to determine frequency and relevance of these mutations in a possibly large series of corticotroph PitNETs. Methods: Study included 147 patients (100 CD and 47 silent tumors) that were screened for hot-spot mutations in USP8, USP48 and BRAF with Sanger sequencing, while 128 of these patients were screened for TP53 mutations with next generation sequencing and immunohistochemistry. Results: USP8 mutations were found in 41% CD and 8,5% silent tumors, while USP48 mutations were found in 6% CD patients only. Both were more prevalent in women. They were related to higher rate of biochemical remission, non-invasive tumor growth, its smaller size and densely granulated histology, suggesting that these mutation may be favorable clinical features. Multivariate survival analyses did not confirm possible prognostic value of mutation in protein deubiquitinases. No BRAF mutations were found. Four TP53 mutations were identified (2 in CD, 2 in silent tumors) in tumors with size >10mm including 3 invasive ones. They were found in Crooke's cell and sparsely granulated tumors. Tumors with missense TP53 mutations had higher TP53 immunoreactivity score than wild-type tumors. Tumor with frameshift TP53 variant had low protein expression. TP53 mutation was a poor prognostic factor in CD according to uni- and multivariate survival analyses in spite of low mutations frequency. Conclusions: We confirmed high prevalence of USP8 mutations and low incidence of USP48 and TP53 mutations. Changes in protein deubiquitinases genes appear to be favorable prognostic factors in CD. TP53 mutations are rare, occur in both functioning and silent tumors and are related to poor clinical outcome in CD.

DNA Methylation Pattern in Somatotroph Pituitary Neuroendocrine Tumors.

INTRODUCTION: Growth hormone secretion by sporadic somatotroph neuroendocrine pituitary tumors (PitNETs) is a major cause of acromegaly. These tumors are relatively heterogenous in terms of histopathological and molecular features. Our previous transcriptomic profiling of somatotroph tumors revealed three distinct molecular subtypes. This study aimed to investigate the difference in DNA methylation patterns in subtypes of somatotroph PitNETs and its role in distinctive gene expression. METHODS: Genome-wide DNA methylation was investigated in 48 somatotroph PitNETs with EPIC microarrays. Gene expression was assessed with RNAseq. Bisulfite pyrosequencing and qRT-PCR were used for verifying the results of DNA methylation and gene expression. RESULTS: Clustering tumor samples based on methylation data reflected the transcriptome-related classification. Subtype 1 tumors are densely granulated without GNAS mutation, characterized by high expression of NR5A1 (SF-1) and GIPR. The expression of both genes is correlated with specific methylation of the gene body and promoter. This subtype has a lower methylation level of 5' gene regions and CpG islands than the remaining tumors. Subtype 2 PitNETs are densely granulated and frequently GNAS-mutated, while those in subtype 3 are mainly sparsely granulated. Methylation/expression analysis indicates that ∼50% genes located in differentially methylated regions are those differentially expressed between tumor subtypes. Correlation analysis revealed DNA methylation-controlled genes, including CDKN1B, CCND2, EBF3, CDH4, CDH12, MGMT, STAT5A, PLXND1, PTPRE, and MMP16, and genes encoding ion channels and semaphorins. CONCLUSION: DNA methylation profiling confirmed the existence of three molecular subtypes of somatotroph PitNETs. High expression of NR5A1 and GIPR in subtype 1 tumors is correlated with specific methylation of both genes.

Epigenetic Downregulation of Hsa-miR-193b-3p Increases Cyclin D1 Expression Level and Cell Proliferation in Human Meningiomas.

Meningiomas are common intracranial tumors in adults. Abnormal microRNA (miRNA) expression plays a role in their pathogenesis. Change in miRNA expression level can be caused by impaired epigenetic regulation of miRNA-encoding genes. We found the genomic region covering the MIR193B gene to be DNA hypermethylated in meningiomas based on analysis of genome-wide methylation (HumanMethylation450K Illumina arrays). Hypermethylation of MIR193B was also confirmed via bisulfite pyrosequencing. Both hsa-miR-193b-3p and hsa-miR-193b-5p are downregulated in meningiomas. Lower expression of hsa-miR-193b-3p and higher MIR193B methylation was observed in World Health Organization (WHO) grade (G) II/III tumors as compared to GI meningiomas. CCND1 mRNA was identified as a target of hsa-miR-193b-3p as further validated using luciferase reporter assay in IOMM-Lee meningioma cells. IOMM-Lee cells transfected with hsa-miR-193b-3p mimic showed a decreased cyclin D1 level and lower cell viability and proliferation, confirming the suppressive nature of this miRNA. Cyclin D1 protein expression (immunoreactivity) was higher in atypical than in benign meningiomas, accordingly to observations of lower hsa-miR-193b-3p levels in GII tumors. The commonly observed hypermethylation of MIR193B in meningiomas apparently contributes to the downregulation of hsa-miR-193b-3p. Since hsa-miR-193b-3p regulates proliferation of meningioma cells through negative regulation of cyclin D1 expression, it seems to be an important tumor suppressor in meningiomas.

DNA methylation, combined with RNA sequencing, provide novel insight into molecular classification of chordomas and their microenvironment.

Chordomas are rare tumors of notochord remnants, occurring mainly in the sacrum and skull base. Despite of their unusually slow growth, chordomas are highly invasive and the involvement of adjacent critical structures causes treatment challenges. Due to the low incidence, the molecular pathogenesis of this entity remains largely unknown. This study aimed to investigate DNA methylation abnormalities and their impact on gene expression profiles in skull base chordomas. 32 tumor and 4 normal nucleus pulposus samples were subjected to DNA methylation and gene expression profiling with methylation microarrays and RNA sequencing. Genome-wide DNA methylation analysis revealed two distinct clusters for chordoma (termed subtypes C and I) with different patterns of aberrant DNA methylation. C Chordomas were characterized by general hypomethylation with hypermethylation of CpG islands, while I chordomas were generally hypermethylated. These differences were reflected by distinct distribution of differentially methylated probes (DMPs). Differentially methylated regions (DMRs) were identified, indicating aberrant methylation in known tumor-related genes in booth chordoma subtypes and regions encoding small RNAs in subtype C chordomas. Correlation between methylation and expression was observed in a minority of genes. Upregulation of TBXT in chordomas appeared to be related to lower methylation of tumor-specific DMR in gene promoter. Gene expression-based clusters of tumor samples did not overlap with DNA methylation-based subtypes. Nevertheless, they differ in transcriptomic profile that shows immune infiltration in I chordomas and up-regulation of cell cycle in C chordomas. Immune enrichment in chordomas I was confirmed with 3 independent deconvolution methods and immunohistochemistry. Copy number analysis showed higher chromosomal instability in C chordomas. Nine out of eight had deletion of CDKN2A/B loci and downregulation of genes encoded in related chromosomal band. No significant difference in patients' survival was observed between tumor subtypes, however, shorter survival was observed in patients with higher number of copy number alterations.

The expression of glucocorticoid and mineralocorticoid receptors in pituitary tumors causing Cushing's disease and silent corticotroph tumors.

Objective: Pituitary neuroendocrine corticotroph tumors commonly cause Cushing's disease (CD) that results from increased adrenocorticotropic hormone (ACTH) secretion by the pituitary tumor and consequent increase of cortisol levels in blood. However, in some patients, corticotroph tumors remain clinically non-functioning. Cortisol secretion is regulated by the hypothalamic-pituitary-adrenal axis and includes a negative feedback between cortisol and ACTH secretion. Glucocorticoids reduce ACTH level both by hypothalamic regulation and acting on corticotrophs via glucocorticoid (GR) and mineralocorticoid (MR) receptors. The aim of the study was to determine the role of GR and MR expression at mRNA and protein levels in both functioning and silent corticotroph tumors. Methods: Ninety-five patients were enrolled, including 70 with CD and 25 with silent corticotroph tumors. Gene expression levels of NR3C1 and NR3C2 coding for GR and MR, respectively, were determined with qRT-PCR in the two tumor types. GR and MR protein abundance was assessed with immunohistochemistry. Results: Both GR and MR were expressed in corticotroph tumors. Correlation between NR3C1 and NR3C2 expression levels was observed. NR3C1 expression was higher in silent than in functioning tumors. In CD patients NR3C1 and NR3C2 levels were negatively correlated with morning plasma ACTH levels and tumor size. Higher NR3C2 was confirmed in patients with remission after surgery and in densely granulated tumors. Expression of both genes and GR protein was higher in USP8-mutated tumors. Similar relationship between USP8 mutations and expression levels were observed in analysis of silent tumors that also revealed a negative correlation between GR and tumor size and higher NR3C1 expression in densely granulated tumors. Conclusions: Although the associations between gene/protein expression and patients clinical features are not strong, they consistently show an evident trend in which higher receptor expression corresponds to more favorable clinical characteristics.

Transcriptomic Classification of Pituitary Neuroendocrine Tumors Causing Acromegaly.

Acromegaly results from growth hormone hypersecretion, predominantly caused by a somatotroph pituitary neuroendocrine tumor (PitNET). Acromegaly-causing tumors are histologically diverse. Our aim was to determine transcriptomic profiles of various somatotroph PitNETs and to evaluate clinical implication of differential gene expression. A total of 48 tumors were subjected to RNA sequencing, while expression of selected genes was assessed in 134 tumors with qRT-PCR. Whole-transcriptome analysis revealed three transcriptomic groups of somatotroph PitNETs. They differ in expression of numerous genes including those involved in growth hormone secretion and known prognostic genes. Transcriptomic subgroups can be distinguished by determining the expression of marker genes. Analysis of the entire cohort of patients confirmed differences between molecular subtypes of tumors. Transcriptomic group 1 includes ~20% of acromegaly patients with GNAS mutations-negative, mainly densely granulated tumors that co-express GIPR and NR5A1 (SF-1). SF-1 expression was verified with immunohistochemistry. Transcriptomic group 2 tumors are the most common (46%) and include mainly GNAS-mutated, densely granulated somatotroph and mixed PitNETs. They have a smaller size and express favorable prognosis-related genes. Transcriptomic group 3 includes predominantly sparsely granulated somatotroph PitNETs with low GNAS mutations frequency causing ~35% of acromegaly. Ghrelin signaling is implicated in their pathogenesis. They have an unfavorable gene expression profile and higher invasive growth rate.

The Expression of Cell Cycle-Related Genes in USP8-Mutated Corticotroph Neuroendocrine Pituitary Tumors and Their Possible Role in Cell Cycle-Targeting Treatment.

Protein deubiquitinases USP8 and USP48 are known driver genes in corticotroph pituitary neuroendocrine tumors (PitNETs). USP8 mutations have pleiotropic effects that include notable changes in genes' expression. Genes involved in cell cycle regulation were found differentially expressed in mutated and wild-type tumors. This study aimed to verify difference in the expression level of selected cell cycle-related genes and investigate their potential role in response to cell cycle inhibitors. Analysis of 70 corticotroph PitNETs showed that USP8-mutated tumors have lower CDKN1B, CDK6, CCND2 and higher CDC25A expression. USP48-mutated tumors have lower CDKN1B and CCND1 expression. A lower p27 protein level in mutated than in wild-type tumors was confirmed that may potentially influence the response to small molecule inhibitors targeting the cell cycle. We looked for the role of USP8 mutations or a changed p27 level in the response to palbociclib, flavopiridol and roscovitine in vitro using murine corticotroph AtT-20/D16v-F2 cells. The cells were sensitive to each agent and treatment influenced the expression of genes involved in cell cycle regulation. Overexpression of mutated Usp8 in the cells did not affect the expression of p27 nor the response to the inhibitors. Downregulating or upregulating p27 expression in AtT-20/D16v-F2 cells also did not affect treatment response.

Difference in miRNA Expression in Functioning and Silent Corticotroph Pituitary Adenomas Indicates the Role of miRNA in the Regulation of Corticosteroid Receptors.

Corticotroph pituitary adenomas commonly cause Cushing's disease (CD), but some of them are clinically silent. The reason why they do not cause endocrinological symptoms remains unclear. We used data from small RNA sequencing in adenomas causing CD (n = 28) and silent ones (n = 20) to explore the role of miRNA in hormone secretion and clinical status of the tumors. By comparing miRNA profiles, we identified 19 miRNAs differentially expressed in clinically functioning and silent corticotroph adenomas. The analysis of their putative target genes indicates a role of miRNAs in regulation of the corticosteroid receptors expression. Adenomas causing CD have higher expression of hsa-miR-124-3p and hsa-miR-135-5p and lower expression of their target genes NR3C1 and NR3C2. The role of hsa-miR-124-3p in the regulation of NR3C1 was further validated in vitro using AtT-20/D16v-F2 cells. The cells transfected with miR-124-3p mimics showed lower levels of glucocorticoid receptor expression than control cells while the interaction between miR-124-3p and NR3C1 3' UTR was confirmed using luciferase reporter assay. The results indicate a relatively small difference in miRNA expression between clinically functioning and silent corticotroph pituitary adenomas. High expression of hsa-miR-124-3p in adenomas causing CD plays a role in the regulation of glucocorticoid receptor level and probably in reducing the effect of negative feedback mediated by corticosteroids.

Invasive and Noninvasive Nonfunctioning Gonadotroph Pituitary Tumors Differ in DNA Methylation Level of LINE-1 Repetitive Elements.

PURPOSE: Epigenetic dysregulation plays a role in pituitary tumor pathogenesis. Some differences in DNA methylation were observed between invasive and noninvasive nonfunctioning gonadotroph tumors. This study sought to determine the role of DNA methylation changes in repetitive LINE-1 elements in nonfunctioning gonadotroph pituitary tumors. METHODS: We investigated LINE-1 methylation levels in 80 tumors and normal pituitary glands with bisulfite-pyrosequencing. Expression of two LINE-1 open reading frames (L1-ORF1 and L1-ORF2) was analyzed with qRT-PCR in tumor samples and mouse gonadotroph pituitary cells treated with DNA methyltransferase inhibitor. Immunohistochemical staining against L1-ORF1p was also performed in normal pituitary glands and tumors. RESULTS: Hypomethylation of LINE-1 was observed in pituitary tumors. Tumors characterized by invasive growth revealed lower LINE-1 methylation level than noninvasive ones. LINE-1 methylation correlated with overall DNA methylation assessed with HM450K arrays and negatively correlated with L1-ORF1 and L1-ORF2 expression. Treatment of αT3-1 gonadotroph cells with 5-Azacytidine clearly increased the level of L1-ORF1 and L1-ORF2 mRNA; however, its effect on LßT2 cells was less pronounced. Immunoreactivity against L1-ORF1p was higher in tumors than normal tissue. No difference in L1-ORF1p expression was observed in invasive and noninvasive tumors. CONCLUSION: Hypomethylation of LINE-1 is related to invasive growth and influences transcriptional activity of transposable elements.

Differential microRNA Expression in USP8-Mutated and Wild-Type Corticotroph Pituitary Tumors Reflect the Difference in Protein Ubiquitination Processes.

BACKGROUND: USP8 mutations are the most common driver changes in corticotroph pituitary tumors. They have direct effect on cells' proteome through disturbance of ubiquitination process and also influence gene expression. The aim of this study was to compare microRNA profiles in USP8-mutated and wild-type tumors and determine the probable role of differential microRNA expression by integrative microRNA and mRNA analysis. METHODS: Patients with Cushing's disease (n = 28) and silent corticotroph tumors (n = 20) were included. USP8 mutations were identified with Sanger sequencing. MicroRNA and gene expression was determined with next-generation sequencing. RESULTS: USP8-mutated patients with Cushing's disease showed higher rate of clinical remission and trend towards lower tumor volume than wild-type patients. Comparison of microRNA profiles of USP8-mutated and wild-type tumors revealed 68 differentially expressed microRNAs. Their target genes were determined by in silico prediction and microRNA/mRNA correlation analysis. GeneSet Enrichment analysis of putative targets showed that the most significantly overrepresented genes are involved in protein ubiquitination-related processes. Only few microRNAs influence the expression of genes differentially expressed between USP8-mutated and wild-type tumors. CONCLUSIONS: Differences in microRNA expression in corticotropinomas stratified according to USP8 status reflect disturbed ubiquitination processes, but do not correspond to differences in gene expression between these tumors.

The Search of miRNA Related to Invasive Growth of Nonfunctioning Gonadotropic Pituitary Tumors.

PURPOSE: Nonfunctioning gonadotropic pituitary neuroendocrine tumors (PitNETs) are among the most frequent neoplasms of pituitary gland. Although PitNETs are commonly considered benign, a notable part of patients suffer from tumor recurrence after treatment. Invasive growth of pituitary tumor is among the most important prognostic factors. Since molecular features of invasiveness are of potential clinical usefulness, this study was aimed to verify whether invasive and noninvasive nonfunctioning gonadotropic PitNETs differ in the miRNA expression profile and whether the differences could provide a possible molecular classifier. METHODS: miRNA profiles were determined in 20 patients (11 invasive and 9 noninvasive tumors) using next-generation sequencing. The expression of selected miRNAs was assessed in the independent cohort of 80 patients with qRT-PCR. RESULTS: When miRNA profiles of invasive and noninvasive tumors were compared, 29 miRNAs were found differentially expressed. Hsa-miR-184, hsa-miR-181a-2-3p, hsa-miR-93-3p, hsa-miR-574-5p, hsa-miR-185-5p, and hsa-miR-3200-5p showed a potential clinical value according to ROC curve analysis. Unfortunately, differential expression of only hsa-miR-185-5p was confirmed in the validation cohort, with AUG at 0.654. CONCLUSION: Differences in miRNAs expression profiles in invasive and noninvasive gonadotropic PitNETs are slight and the level of miRNA expression seems not to be applicable as useful classifier of tumor invasiveness.

DNA Methylation Influences miRNA Expression in Gonadotroph Pituitary Tumors.

microRNAs are involved in pathogenesis of cancer. DNA methylation plays a role in transcription of miRNA-encoding genes and may contribute to changed miRNA expression in tumors. This issue was not investigated in pituitary neuroendocrine tumors (PitNETs) previously. DNA methylation patterns, assessed with HumanMethylation450K arrays in 34 PitNETs and five normal pituitaries, were used to determine differentially methylated CpGs located at miRNA genes. It showed aberrant methylation in regions encoding for 131 miRNAs. DNA methylation data and matched miRNA expression profiles, determined with next-generation sequencing (NGS) of small RNAs, were correlated in 15 PitNETs. This showed relationship between methylation and expression levels for 12 miRNAs. DNA methylation and expression levels of three of them (MIR145, MIR21, and MIR184) were determined in the independent group of 80 tumors with pyrosequencing and qRT-PCR and results confirmed both aberrant methylation in PitNETs and correlation between methylation and expression. Additionally, in silico target prediction was combined with analysis of established miRNA profiles and matched mRNA expression pattern, assessed with amplicon-based NGS to indicate putative target genes of epigenetically deregulated miRNAs. This study reveals aberrant DNA methylation in miRNA-encoding genes in gonadotroph PitNETs. Methylation changes affect expression level of miRNAs that regulate putative target genes with tumorigenesis-relevant functions.

The Role of Aberrant DNA Methylation in Misregulation of Gene Expression in Gonadotroph Nonfunctioning Pituitary Tumors.

Gonadotroph nonfunctioning pituitary adenomas (NFPAs) are common intracranial tumors, but the role of aberrant epigenetic regulation in their development remains poorly understood. In this study, we investigated the effect of impaired CpG methylation in NFPAs. We determined DNA methylation and transcriptomic profiles in 32 NFPAs and normal pituitary sections using methylation arrays and sequencing, respectively. Ten percent of differentially methylated CpGs were correlated with gene expression, and the affected genes are involved in a variety of tumorigenesis-related pathways. Different proportions of gene body and promoter region localization were observed in CpGs with negative and positive correlations between methylation and gene expression, and different proportions of CpGs were located in 'open sea' and 'shelf/shore' regions. The expression of ~8% of genes differentially expressed in NFPAs was related to aberrant methylation. Methylation levels of seven CpGs located in the regulatory regions of FAM163A, HIF3A and PRSS8 were determined by pyrosequencing, and gene expression was measured by qRT-PCR and immunohistochemistry in 83 independent NFPAs. The results clearly confirmed the negative correlation between methylation and gene expression for these genes. By identifying which aberrantly methylated CpGs affect gene expression in gonadotrophinomas, our data confirm the role of aberrant methylation in pathogenesis of gonadotroph NFPAs.

USP8 mutations in corticotroph adenomas determine a distinct gene expression profile irrespective of functional tumour status.

OBJECTIVE: Pituitary corticotroph adenomas commonly cause Cushing's disease (CD) but part of these tumours are hormonally inactive (silent corticotroph adenomas, SCA). USP8 mutations are well-known driver mutations in corticotrophinomas. Differences in transcriptomic profiles between functioning and silent tumours or tumours with different USP8 status have not been investigated. DESIGN AND METHODS: Forty-eight patients (28 CD, 20 SCA) were screened for USP8 mutations with Sanger sequencing. Twenty-four patients were included in transcriptomic profiling with Ampliseq Transcriptome Human Gene Expression Core Panel. The entire patients group was included in qRT-PCR analysis of selected genes expression. Immunohistochemistry was used for visualization of selected protein. RESULTS: We found USP8 mutation in 15 patients with CD and 4 SCAs. USP8 mutations determine molecular profile of the tumours as showed by hierarchical clustering and identification of 1648 genes differentially expressed in USP8-mutated and USP8-wild-type tumours. Mutations affect many molecular pathways as observed in Gene Set Enrichment analysis. USP8-mutated adenomas showed higher level of POMC, CDC25A, MAPK4 but lower level of CCND2, CDK6, CDKN1B than USP8-wt tumours. Eighty-seven genes differentially expressed between CD-related adenomas and SCAs were found, including those involved in cell signalling (GLI2, DLC1, TBX2, RASSF6), cell adhesion (GJA1, CDH6), ion transport (KCNN4, KCNJ5) and GABA signalling (GABBR2, GABRD). CONCLUSION: USP8 mutations occur in functioning and silent corticotrophinomas. They have pleiotropic effect, not limited to EGFR signalling, and affect expression levels of many genes involved in different pathways. Expression of GABA-related genes GABBR2, GNAL, GABARD and KCNJ5 correspond to functional status of the tumours.

Telomere length and TERT abnormalities in pituitary adenomas.

OBJECTIVES: Pituitary adenomas (PAs) are among the most frequent intracranial tumors in humans. Abnormal telomerase activity and telomere lengthening are features of tumor cells. They may result from mutations in TERT promoter region, gene amplification or aberrant DNA methylation pattern. Such changes were found in variety of tumors including those of brain. Aim of the study was to evaluate the incidence of TERT abnormalities and to assess their role in telomere lengthening in PAs. METHODS: Study involved 101 patients with PA including both nonfunctioning and functioning subtypes. Telomerase length as well as TERT mRNA level and gene amplification were estimated using quantitative PCR (qPCR). Promoter mutations were assessed using Sanger sequencing. The results from genome-wide DNA methylation profiling with HumanMethylation 450K (Illumina) were used for the analysis of TERT locus. RESULTS: Variable telomere length was observed in patients, however no relationship with clinicopathological features was found. We observed a missense variant in TERT promoter in one patient only whereas increased TERT copy number were identified in 6 patients (5.6%). However no relationship between these results and telomere length or TERT expression was found. DNA methylation at TERT locus was not found to be changed when adenoma samples and normal tissue sections were compared. CONCLUSION: The results indicate that telomerase abnormalities do not play a role in pathogenesis of pituitary tumors.

DNA methylation profiling in nonfunctioning pituitary adenomas.

Nonfunctioning pituitary adenomas (NFPAs) are among the most frequent intracranial tumors but their molecular background, including changes in epigenetic regulation, remains poorly understood. We performed genome-wide DNA methylation profiling of 34 NFPAs and normal pituitary samples. Methylation status of the selected genomic regions and expression level of corresponding genes were assessed in a group of 75 patients. NFPAs exhibited distinct global methylation profile as compared to normal pituitary. Aberrant DNA methylation appears to contribute to deregulation of the cancer-related pathways as shown by preliminary functional analysis. Promoter hypermethylation and decreased expression level of SFN, STAT5A, DUSP1, PTPRE and FGFR2 was confirmed in the enlarged group of NFPAs. Difference in the methylation profiles between invasive and non-invasive NFPAs is very slight. Nevertheless, invasiveness-related aberrant epigenetic deregulation of the particular genes was found including upregulation of ITPKB and downregulation CNKSR1 in invasive tumors.

Downregulation of PTPRH (Sap-1) in colorectal tumors.

Tyrosine phosphorylation is one of the basic mechanisms for signal transduction in the cell. Receptors exhibiting tyrosine kinase activity are widely involved in carcinogenesis and are negatively regulated by receptor protein tyrosine phosphatases (RPTP). Genes encoding different RPTPs are affected by aberrant epigenetic regulation in cancer. PTPRH (SAP-1) has been previously described to be overexpressed in colorectal cancer (CRC) and classified as an oncogenic factor. Previous microarray-based mRNA expression comparison of colorectal adenomas (AD), CRC and normal mucosa samples (NM) demonstrated that PTPRH tumor expression is the most reduced of all RPTP genes. qRT-PCR validation revealed gene downregulation for CRC (7.6-fold-change; P<0.0001) and AD (3.4-fold-change; P<0.0001) compared to NM. This was confirmed by immunohistochemical staining of tumor and NM sections as pronounced decrease of protein expression was observed in CRCs compared to the corresponding normal tissue. DNA methylation of two PTPRH promoter fragments was analyzed by pyrosequencing in a group of CRC, and AD patients as well as NM samples and CRC cell lines. The mean DNA methylation levels of these two regions were significantly higher in CRC than in NM. Both regions were highly methylated in SW480 and HCT116 cell lines contrary to unmethylated HT29 and COLO205. Cell lines with highly methylated promoters notably showed lower PTPRH expression levels, lower RNA II polymerase concentrations and higher levels of H3K27 trimethylation in the promoter and gene body, measured by chromatin immunoprecipitation. Cells were cultured with 5-aza-deoxycitidine and an increase in PTPRH expression was observed in SW480 and HCT116, whereas this was unchanged in the unmethylated cell lines. The results indicate that PTPRH is downregulated in colorectal tumors and its expression is epigenetically regulated via DNA methylation and chromatin modifications.

Mutational Analysis of Recurrent Meningioma Progressing From Atypical to Rhabdoid Subtype.

BACKGROUND: Rhabdoid meningioma is rare aggressive meningioma histological subtype that develops predominantly through progression from less malignant tumors. Owing to its low incidence, this tumor's biological background is unknown. The aim of this study was to profile somatic mutations in 4 meningioma samples from the same patient, derived previously from 4 subsequent tumor resections. CASE DESCRIPTION: A 58-year-old woman presented with recurrent meningioma progressing from atypical to rhabdoid subtype. Four tumor samples that represent a primary tumor (atypical GII) and 3 recurrent tumors that were subsequently removed (anaplastic GIII, rhabdoid GIII, and anaplastic/rhabdoid GIII) from this patient were subjected to mutational analysis of coding sequences of 952 tumor-related genes. Three mutations were identified in all tumor samples exhibiting a high allelic frequency: ARID1A frameshift deletion, NF2 in-frame deletion, and missense variant of SRSF2. The predicted inactivating effect of ARID1A deletion was confirmed by immunohistochemical staining of tumor sections in which a high proportion of cells lacked protein expression. Additional low-allelic-fraction mutations were observed in all tumor samples, likely representing "passenger," low-effect mutations that reflect a clonal selection of tumor cells through malignant progression of the meningioma. CONCLUSION: The mutation of ARID1A that encodes the subunit of the SWI/SNF complex represents the most likely driver of the tumor's malignant potential. It also may be involved in the acquisition of the rhabdoid phenotype, given that mutations in chromatin remodeling proteins are the hallmark of atypical teratoid/rhabdoid tumors.

Microsatellite instability analysis in pituitary adenomas.

OBJECTIVE: Mutator phenotypes with microsatellite instability (MSI) are observed in a subset of solid tumors including those localized in the brain. MSI arises from impaired DNA mismatch repair. It can be a potential marker of resistance to radiation and chemotherapy, as demonstrated for several cancer types. Our study aims are to investigate MSI incidence in pituitary adenomas (PA) with a currently recommended methodology. METHODS: DNA was obtained from 107 patients with PA of which 83 adenomas were nonfunctioning, 13 somatotrophic, 9 lactotrophic and 2 corticotrophic. These were examined for MSI status by PCR and capillary electrophoresis using five quasimonomorphic microsatellite markers: BAT25, BAT26, NR21, NR24 and NR27; in accordance to current Bethesda guidelines. RESULTS AND CONCLUSION: No microsatellite instability was detected in the tumor samples thus implying the lack of any clinical usefulness of MSI testing in PA cases.