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.

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.

Functional status of surgically treated pineal cyst patients.

BACKGROUND: Microsurgical removal represents a well-accepted treatment option for symptomatic benign pineal cysts (PCs). However, very few studies have quantitatively evaluated the functional status of surgically treated PC patients. METHODS: A detailed analysis of preoperative, immediate postoperative, and long-term clinical and radiological characteristics was performed. The functional status of the patients was categorized using the modified Rankin scale (mRS) and the Chicago Chiari Outcome Scale (CCOS). In addition, a comparative analysis between pediatric and adult patients with PCs was carried out. RESULTS: Overall, pediatric patients experienced better long-term mRS scores than adults. The differences between the pre-, the immediate post-, and the last postoperative mRS of the patients were statistically significant for the total population (P < 0.001). All patients obtained a CCOS of 11 or more, which reflects a good/optimal result after microneurosurgery. The type of the surgical approach was independently associated with the postoperative complications (P < 0.01), more frequently reported with the midline supracerebellar infratentorial (SCIT) approach than with its paramedian modification. CONCLUSION: The functional status of properly selected symptomatic patients with PCs may improve significantly after their surgical management through a paramedian SCIT approach in sitting position.

Long-term survival outcomes of pineal region gliomas.

PURPOSE: Surgical series of pineal region gliomas are rarely available. Whereas it is a general assumption that the extent of surgical resection correlates with survival outcomes of intracranial gliomas; the impact of the microsurgical resection on the long-term outcomes of pineal gliomas has been questioned. We present a surgical series of pineal region gliomas with focus on the survival outcome analysis. METHODS: 17 histologically confirmed pineal region glioma patients classified as diffuse and non-diffuse gliomas were retrospectively analyzed. A detailed description of the series was followed by regression models to identify predictors of clinical outcomes. Uni- a multivariate survival analysis was performed to determine independent predictors of mortality. RESULTS: Although the number of treated patients was small, only WHO grade histopathology remained significant (p = 0.02) after multivariate survival analysis with extent of resection, age, tumor volume, and preoperative functional status. The extent of the surgical resection did not correlate with the disease survival rates of non-diffuse (p = 1), diffuse (p = 0.2), nor all gliomas (p = 0.6). 15 of 17 patients underwent gross total (nine patients) or subtotal resection. The preoperative functional status of the patients showed overall improvement on the immediate (p < 0.001) and long-term (p = 0.03) follow-up after 106 (3 - 324) months. CONCLUSION: The extent of the surgical resection does not seem to significantly impact on the survival outcomes of pineal region gliomas. Thus, genotype and molecular features may essentially affect the outcome. Further research on the field is required.

Systematic review of pineal cysts surgery in pediatric patients.

INTRODUCTION: We present a consecutive case series and a systematic review of surgically treated pediatric PCs. We hypothesized that the symptomatic PC is a progressive disease with hydrocephalus at its last stage. We also propose that PC microsurgery is associated with better postoperative outcomes compared to other treatments. METHODS: The systematic review was conducted in PubMed and Scopus. No clinical study on pediatric PC patients was available. We performed a comprehensive evaluation of the available individual patient data of 43 (22 case reports and 21 observational series) articles. RESULTS: The review included 109 patients (72% females). Ten-year-old or younger patients harbored smaller PC sizes compared to older patients (p < 0.01). The pediatric PCs operated on appeared to represent a progressive disease, which started with unspecific symptoms with a mean cyst diameter of 14.5 mm, and progressed to visual impairment with a mean cyst diameter of 17.8 mm, and hydrocephalus with a mean cyst diameter of 23.5 mm in the final stages of disease (p < 0.001). Additionally, 96% of patients saw an improvement in their symptoms or became asymptomatic after surgery. PC microsurgery linked with superior gross total resection compared to endoscopic and stereotactic procedures (p < 0.001). CONCLUSIONS: Surgically treated pediatric PCs appear to behave as a progressive disease, which starts with cyst diameters of approximately 15 mm and develops with acute or progressive hydrocephalus at the final stage. PC microneurosurgery appears to be associated with a more complete surgical resection compared to other procedures.

Supportive roles of brain macrophages in CNS metastases and assessment of new approaches targeting their functions.

Metastases to the central nervous system (CNS) occur frequently in adults and their frequency increases with the prolonged survival of cancer patients. Patients with CNS metastases have short survival, and modern therapeutics, while effective for extra-cranial cancers, do not reduce metastatic burden. Tumor cells attract and reprogram stromal cells, including tumor-associated macrophages that support cancer growth by promoting tissue remodeling, invasion, immunosuppression and metastasis. Specific roles of brain resident and infiltrating macrophages in creating a pre-metastatic niche for CNS invading cancer cells are less known. There are populations of CNS resident innate immune cells such as: parenchymal microglia and non-parenchymal, CNS border-associated macrophages that colonize CNS in early development and sustain its homeostasis. In this study we summarize available data on potential roles of different brain macrophages in most common brain metastases. We hypothesize that metastatic cancer cells exploit CNS macrophages and their cytoprotective mechanisms to create a pre-metastatic niche and facilitate metastatic growth. We assess current pharmacological strategies to manipulate functions of brain macrophages and hypothesize on their potential use in a therapy of CNS metastases. We conclude that the current data strongly support a notion that microglia, as well as non-parenchymal macrophages and peripheral infiltrating macrophages, are involved in multiple stages of CNS metastases. Understanding their contribution will lead to development of new therapeutic strategies.

Midline and Paramedian Supracerebellar Infratentorial Approach to The Pineal Region: A Comparative Clinical Study in 112 Patients.

OBJECTIVE: The midline supracerebellar infratentorial (SCIT) approach and its paramedian development are commonly used for dealing with pineal lesions. Comparative clinical studies are lacking, however. We aim to establish the better performance of the paramedian SCIT approach in terms of clinical safety in surgically treated pineal cysts and pineal region tumors. Procedural functionality and effectiveness have been also analyzed. METHODS: A comparative analysis of clinical, radiologic, pathologic, and surgical features, and outcome was performed between 55 midline and 57 paramedian SCIT approaches that were exclusively performed in 112 patients (57 pineal cysts and 55 tumors of the pineal region) operated in sitting position by a single surgeon. Information was retrieved from hospital records and microsurgical videos. RESULTS: The paramedian SCIT approach linked with fewer postoperative complications (odds ratio [OR]: 0.40) and fewer approach-related complications (OR: 0.28) than the midline SCIT approach. The SCIT paramedian approach was achieved in a shorter time, by a smaller bone flap, and with fewer complex procedural steps than the midline approach. The SCIT paramedian approach did not require the opening of the falx cerebelli, midline cerebellar retraction, section of the midline cerebellar draining veins, nor wide opening of the dura. Gross total resection, size of the lesion, microsurgical time for removal, histopathological diagnosis and postoperative outcome were statistically similar in both groups. CONCLUSIONS: The SCIT approach represents a safer and more functional approach for the removal of cysts and tumors of the pineal region than the classic midline approach, while maintaining the same effectiveness.

Pineal Gland Tumor Microenvironment.

A wide and heterogeneous variety of tumors develop from the pineal gland. Pineal parenchymal tumors, germ cell tumors, and glial tumors represent most of them. The molecular profiles and tumor microenvironment play a key role in the development and progression of pineal gland tumors. Consequently, they represent important factors that may determine the efficacy of the different treatment modalities and the clinical outcome. Current literature is scarce regarding the microenvironment research of pineal gland tumors. Here, we review the cellular and molecular profile of the pineal gland tumor microenvironment.

Immune Microenvironment of Brain Metastases-Are Microglia and Other Brain Macrophages Little Helpers?

Brain metastases are common intracranial neoplasms and their frequency increases with prolonged survival of cancer patients. New pharmaceuticals targeting oncogenic kinases and immune checkpoint inhibitors augment both overall and progression-free survival in patients with brain metastases, but are not fully successful in reducing metastatic burden and still a majority of oncologic patients die due to dissemination of the disease. Despite therapy advancements, median survival of patients with brain metastases is several months, although it may vary in different types or subtypes of cancer. Contribution of the innate immune system to cancer progression is well established. Tumor-associated macrophages (TAMs), instead of launching antitumor responses, promote extracellular matrix degradation, secrete immunosuppressive cytokines, promote neoangiogenesis and tumor growth. While their roles as pro-tumorigenic cells facilitating tissue remodeling, invasion and metastasis is well documented, much less is known about the immune microenvironment of brain metastases and roles of specific immune cells in those processes. The central nervous system (CNS) is armed in resident myeloid cells: microglia and perivascular macrophages which colonize CNS in early development and maintain homeostasis in brain parenchyma and at brain-blood vessels interfaces. In this study we discuss available data on the immune composition of most common brain metastases, focusing on interactions between metastatic cancer cells and microglia, perivascular and meningeal macrophages. Cancer cells 'highjack' several CNS protective mechanisms and may employ microglia and CNS-border associated macrophages into helping cancer cells to colonize a pre-metastatic niche. We describe emerging molecular insights into mechanisms governing communication between microglia and metastatic cancer cells that culminate in activation of CNS resident microglia and trafficking of monocytic cells from the periphery. We present mechanisms controlling those processes in brain metastases and hypothesize on potential therapeutic approaches. In summary, microglia and non-parenchymal brain macrophages are involved in multiple stages of a metastatic disease and, unlike tumor cells, are genetically stable and predictable, which makes them an attractive target for anticancer therapies.

Clinical and immunological correlates of long term survival in glioblastoma.

Glioblastoma (GBM) is the most common and most aggressive type of primary brain tumour in adults. It represents 54% of all gliomas and 16% of all brain tumours (Ostrom et al. 2016). Despite surgery and treatment with radiotherapy plus an oral alkylating agent, temozolomide (TMZ), tumours invariably recur, and the patient survival is an average of ~14-16 months. In this review we summarise the current understanding of multiple factors that may affect survival of patients with GBMs. In particular, we discuss recent advancements in surgery and detection of genomic-based markers with prognostic values, such as IDH1/2 mutations, MGMT gene promoter methylation, and TERT gene promoter alterations. We address the issue of tumour heterogeneity and evolution that may result in different parts of the same tumour exhibiting different GBM subtypes and in subtype switching, which may restrict the usefulness of the expression-based classification as a prognostic marker before relapse. The determinants of long-term survival in patients with IDH1/2wt GBM, beyond MGMT promoter methylation, remain to be identified, and even the absence of both IDH1/2 mutations and MGMT promoter methylation does not preclude long-term survival. These findings suggest that host-derived factors, such as immune system responsiveness may contribute to long-term survival in such patients. We report the results of high-throughput approaches, suggesting links between long-term survival and enhanced immune-related gene expression. The further search for new gene candidates, promoter methylation status, and specific features of host immunity should provide prognostic biomarkers for the evaluation of survival of IDH1 wild-type/non-G-CIMP GBMs.