Glioblastoma epigenomics discloses a complex biology and potential therapeutic targets.

Background and purpose:

Glioblastoma (GBM), a highly aggressive form of brain tumors, has been extensively studied using OMICS methods, and the most characteristic molecular determinants have been incorporated into the histopathological diagnosis. Research data, nevertheless, only partially have been adopted in clinical practice. Here we aimed to present results of our epige­no­mic GBM profiling to better understand early and late determinants of these tumors, and to share main elements of our findings with practicing professionals.

GBM specimens were surgically obtained after first diagnosis (GBM1) and at recurrence (GBM2). DNA was extracted from 24 sequential pairs of formalin-fixed, paraffin-embedded tumor tissues. The Reduced Representation Bisulfite Sequencing kit was used for library preparation. Pooled libraries were sequenced on an Illumina NextSeq 550 instrument. Methylation controls (MC) were obtained from a publicly available database. Bioinformatic analyses were performed to identify differentially methylated pathways and their elements in cohorts of MC, GBM1 and GBM2.

Several differentially methylated pathways involved in basic intracellular and brain tissue developmental processes were identified in the GBM1 vs. MC and GBM2 vs. MC comparisons. Among differentially me­thylated pathways, those involved in immune regulation, neurotransmitter (particularly dopaminergic, noradrenergic and glutaminergic) responses and regulation of stem cell differentiation and proliferation stood out in the GBM2 vs. GBM1 comparisons.

Our study revealed biological complexity of early and late gliomagenesis encompassing mechanisms from basic intracellular through distorted neurodevelopmental processes to more specific immune and highjacked neurotransmitter pathways in the tumor microenvironment. These findings may offer considerations for therapeutic approaches.

Quantitative Analysis of NKX2-3 Expression in Human Colon: An Immunohistochemical Study.

In mice, Nkx2-3 homeodomain transcription factor defines the vascular specification of secondary and tertiary lymphoid tissues of the intestines. In human studies, polymorphisms in NKX2-3 have been identified as a susceptibility factor in inflammatory bowel diseases, whereas in mice, its absence is associated with protection against experimental colitis and enhanced intestinal epithelial proliferation. Here, we investigated the expression of NKX2-3 in normal, polyp, and adenocarcinoma human colon samples using immunohistochemistry and quantitative morphometry, correlating its expression with endothelial and mesenchymal stromal markers. Our results revealed that the expression of NKX2-3 is regionally confined to the lamina propria and lamina muscularis mucosae, and its production is restricted mostly to endothelial cells and smooth muscle cells with variable co-expression of CD34, alpha smooth muscle antigen (αSMA), and vascular adhesion protein-1 (VAP-1). The frequency of NKX2-3-positive cells and intensity of expression correlated inversely with aging. Furthermore, in most colorectal carcinoma samples, we observed a significant reduction of NKX2-3 expression. These findings indicate that the NKX2-3 transcription factor is produced by both endothelial and non-endothelial tissue constituents in the colon, and its expression changes during aging and in colorectal malignancies. (J Histochem Cytochem XX: XXX-XXX, XXXX).

Low-burden TP53 mutations represent frequent genetic events in CLL with an increased risk for treatment initiation.

TP53 aberrations predict chemoresistance and represent a contraindication for the use of standard chemoimmunotherapy in chronic lymphocytic leukaemia (CLL). Recent next-generation sequencing (NGS)-based studies have identified frequent low-burden TP53 mutations with variant allele frequencies below 10%, but the clinical impact of these low-burden TP53 mutations is still a matter of debate. In this study, we aimed to scrutinise the subclonal architecture and clinical impact of TP53 mutations using a sensitive, NGS-based mutation analysis in a 'real-world' cohort of 901 patients with CLL. In total, 225 TP53 mutations were identified in 17.5% (158/901) of the patients; 48% of these alterations represented high-burden mutations, while 52% were low-burden TP53 mutations. Low-burden mutations as sole alterations were identified in 39% (62/158) of all mutated cases with 82% (51/62) of these being represented by a single low-burden TP53 mutation. Patients harbouring low-burden TP53 mutations had significantly lower time to first treatment compared to patients with wild-type TP53. Our study has expanded the knowledge on the frequency, clonal architecture, and clinical impact of low-burden TP53 mutations. By demonstrating that patients with sole low-burden TP53 variants represent more than one-third of patients with TP53 mutations and have an increased risk for treatment initiation, our findings strengthen the need to redefine the threshold of TP53 variant reporting to below 10% in the routine diagnostic setting.

[Practical approach to molecular pathology of central nervous system tumours]. / A központi idegrendszeri daganatok molekuláris patológiája gyakorlati szempontból.

Recent advances in molecular diagnostics are transforming the classification of malignant tumours, it has long played a major role in the field of CNS tumours. Examination of 1p/19q codeletion is indispensable in case of diffuse gliomas. Glioblastoma may be diagnosed even in the absence of characteristic morphological features, when EGFR amplification, TERT promoter mutation or +7/-10 copy number abnormalities are present. The number of entities defined by a genetic abnormality is growing. Comprehensive analysis of DNA methylation may be of considerable help in addition to histology and basic molecular studies, especially in case of small samples. Keeping up with the ever-expanding diagnostic repertoire is difficult, however, advantages and disadvantages of these methods and the context in which they may be useful should be understood by those who are involved in the diagnosis of CNS tumours. This summary provides a general overview of the main methods used in molecular diagnostics.

Clinicopathological analysis of diffuse large B-cell lymphoma using molecular biomarkers: a retrospective analysis from 7 Hungarian centers.

Background: The clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy. Methods: This is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression. Results: A total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4-62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS. Conclusion: In our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.

PersonALL: a genetic scoring guide for personalized risk assessment in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. METHODS: Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. RESULTS: CNAs were detected in 93.8% (n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status (IKZF1normal, IKZF1del and IKZF1plus) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low (n = 215): 86.3%, IKAROS-medium (n = 27): 57.4% and IKAROS-high (n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients (n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients (n = 103) showed high (n = 74) or ultra-poor (n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). CONCLUSIONS: PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

Parallel testing of liquid biopsy (ctDNA) and tissue biopsy samples reveals a higher frequency of EZH2 mutations in follicular lymphoma.

BACKGROUND: Recent genomic studies revealed enhancer of zeste homolog 2 (EZH2) gain-of-function mutations, representing novel therapeutic targets in follicular lymphoma (FL) in around one quarter of patients. However, these analyses relied on single-site tissue biopsies and did not investigate the spatial heterogeneity and temporal dynamics of these alterations. OBJECTIVES: We aimed to perform a systematic analysis of EZH2 mutations using paired tissue (tumor biopsies [TB]) and liquid biopsies (LB) collected prior to treatment within the framework of a nationwide multicentric study. METHODS: Pretreatment LB and TB samples were collected from 123 patients. Among these, 114 had paired TB and LB, with 39 patients characterized with paired diagnostic and relapse samples available. The EZH2 mutation status and allele burden were assessed using an in-house-designed, highly sensitive multiplex droplet digital PCR assay. RESULTS: EZH2 mutation frequency was found to be 41.5% in the entire cohort. In patients with paired TB and LB samples, EZH2 mutations were identified in 37.8% of the patients with mutations exclusively found in 5.3% and 7.9% of TB and LB samples, respectively. EZH2 mutation status switch was documented in 35.9% of the patients with paired diagnostic and relapse samples. We also found that EZH2 wild-type clones may infiltrate the bone marrow more frequently compared to the EZH2 mutant ones. CONCLUSION: The in-depth spatio-temporal analysis identified EZH2 mutations in a considerably higher proportion of patients than previously reported. This expands the subset of FL patients who most likely would benefit from EZH2 inhibitor therapy.

Next-Generation Sequencing-Based Genomic Profiling of Children with Acute Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality, with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML Berlin-Frankfurt-Münster protocols. Targeted next-generation sequencing of 54 genes revealed 17 genes that were recurrently mutated in >5% of patients. Considerable differences were observed in the mutational profiles compared with previous studies, as BCORL1, CUX1, KDM6A, PHF6, and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in the BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, and WT1) mutations were found to be associated with induction failure and shorter event-free survival, suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% versus 44.4%) and transcription factors (35.1% versus 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identify previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.

Epigenetic Suppression of the IL-7 Pathway in Progressive Glioblastoma.

BACKGROUND: Immune evasion in glioblastoma (GBM) shields cancer cells from cytotoxic immune response. METHODS: We investigated CpG methylation in promoters, genes, and pathways in 22 pairs of formalin-fixed paraffin-embedded sequential (FFPE) GBM using restricted resolution bisulfite sequencing (RRBS) and bioinformatic analyses. RESULTS: Gene ontology revealed hypermethylation in elements of the innate and adaptive immune system when recurrent GBM samples (GBMrec) were compared to control (CG) and primary GBM samples (GBMprim). Higher methylation levels of the IL-7 signaling pathway and response to IL-7 were found in GBMrec suggesting a progressive blockade of the IL-7 driven T cell response in sequential GBM. Analyses of the Cancer Genome Atlas array-based data confirmed hypermethylation of the IL-7 pathway in recurrent compared with primary GBM. We also quantified DNA CpG methylation in promoter and gene regions of the IL-7 ligand and IL-7 α-receptor subunit in individual samples of a large RRBS-based sequential cohort of GBM in a Viennese database and found significantly higher methylation levels in the IL-7 receptor α-subunit in GBMrec compared with GBMprim. CONCLUSIONS: This study revealed the progressive suppression of the IL-7 receptor-mediated pathway as a means of immune evasion by GBM and thereby highlighted it as a new treatment target.

[Case report: molecular analysis of congenital glioblastoma in a newborn]. / Esettanulmány: molekuláris vizsgálatok újszülöttkori glioblasztómában.

BACKGROUND: Congenital glioblastoma (cGBM) is a brain tumor very rarely observed in newborns and young infants, and differs in several respects from glioblastoma (GBM) of childhood and adulthood. Our aim was the presentation of a cGBM case with 14 days of postnatal survival at the Pediatric Oncology Center of the Markusovszky University Teaching Hospital in 2004. We investigated formalin-fixed, paraffin-embedded autoptic tumor samples of the newborn by immunohistochemical and molecular genetic (FISH and pyrosequencing) methods. We found polysomy of chromosome 2 and 5' deletion of the ALK gene in the glioma cells by ALK FISH. This result indicates the importance of molecular analyses in the diagnostic evaluation of cGBM, and raises the possibility of a personalized, targeted therapy (crizotinib, alectinib).

Distinct miRNA Expression Signatures of Primary and Secondary Central Nervous System Lymphomas.

Central nervous system (CNS) lymphoma is a rare and aggressive non-Hodgkin lymphoma that might arise in the CNS (primary CNS lymphoma) or disseminates from a systemic lymphoma to the CNS (secondary CNS lymphoma). Dysregulated expression of miRNAs is associated with various pathologic processes, and miRNA expression patterns may have diagnostic, prognostic, and therapeutic implications. However, miRNA expression is understudied in CNS lymphomas. We performed expression analysis of 798 miRNAs in 73 CNS lymphoma samples using the NanoString platform, followed by an analysis to identify potential diagnostic biomarkers characterizing subgroups and to examine differences based on their primary and secondary nature, molecular subtype, mutational patterns, and survival. Thirty-one differentially expressed miRNAs were identified between primary and secondary groups. In addition, 7 more miRNAs were identified associated with a molecular subtype and 25 associated with mutation status. Using unsupervised clustering methods, a small but distinct primary CNS lymphoma subgroup, with characteristically different expression patterns compared with the rest of the cases was defined. Finally, differentially regulated pathways were identified in the above comparisons and the utility of miRNA expression patterns in predicting survival was assessed. Our study identifies a novel CNS lymphoma subgroup defined by distinct miRNAs, proves the importance of specific miRNAs and pathways in the pathogenesis of CNS lymphomas, and provides the basis for future research in defining potential biomarkers.

O-GlcNAcylation in early stages of chronic lymphocytic leukemia: Protocol development for flow cytometry.

BACKGROUND: Recent studies proved that metabolic changes in malignant disorders have an impact on protein glycosylation, however, only a few attempts have been made so far to use O-GlcNAc analysis as a prognostic tool. Glucose metabolism is reported to be altered in hematological malignancies thus, we hypothesized that monitoring intracellular O-GlcNAc levels in Rai stage 0-I (Binet A) CLL patients could give deeper insights regarding subtle metabolic changes of progression which are not completely detected by the routine follow-up procedures. OBJECTIVE: In this proof of concept study we established a flow cytometric detection method for the assessment of O-GlcNAcylation as a possible prognostic marker in CLL malignancy which was supported by fluorescence microscopy. METHODS: Healthy volunteers and CLL patients were recruited for this study. Lymphocytes were isolated, fixed and permeabilised by various methods to find the optimal experimental condition for O-GlcNAc detection by flow cytometry. O-GlcNAc levels were measured and compared to lymphocyte count and various blood parameters including plasma glucose level. RESULTS: The protocol we developed includes red blood cell lysis, formalin fixation, 0.1% Tween 20 permeabilisation and employs standardized cell number per sample and unstained controls. We have found significant correlation between O-GlcNAc levels and WBC (R2= 0.8535, p< 0.0029) and lymphocyte count (R2= 0.9225, p< 0.0006) in CLL patients. Interestingly, there was no such correlation in healthy individuals (R2= 0.05664 for O-GlcNAc vs WBC and R2= 0.04379 for O-GlcNAc vs lymphocytes). CONCLUSION: Analyzing O-GlcNAc changes in malignant disorders, specifically in malignant hematologic diseases such as CLL, could be a useful tool to monitor the progression of the disease.

[A case of destructive cervical spondylarthropathy related to chronic dialysis]. / Krónikus dialízis kapcsán kialakult destruktív cervicalis spondylarthropathia esete.

A case of a 61-year-old male patient suffered chronic renal failure and dialysed for 23 years with destructive cervical spondylarthropathy is presented. The patient presented with sudden onset of cervical pain radiating into his shoulders without neurological deficits. CT and MRI of the cervical and thoracic spine revealed severe destructive changes and compressive fractures of C6 and C7 vertebrae which caused the narrowing of the nerve root canals at these levels. A 360-degree fixation was performed to treat the unstable fracture and the patient's pain (C6 and C7 corpectomy, autolog bone graft replacement of the two vertebral bodies, anterior plate fixation and posterior instrumentation with screws and rods). Postoperatively the patient had no significant pain, no neurological deficit and he was able to manage independent life himself. During the immediate follow-up CT of the neck showed the satisfactory position of the bone graft and the metal implantations. The 6 months follow-up CT revealed the anterior migration of the two screws from the Th1 vertebral body and 2 mm ventral elevation of the caudal end of the plate from the anterior surface of the Th1 vertebral body. The 1-year follow-up could not be performed because the patient died due to cardio-pulmonary insufficiency. This is the second Hungarian report of a chronic dialysis related severe spondylarthropathy which may cause pathologic fractures of the vertebral bodies. The typical radiological and histological findings are discussed. This disease affect patients' quality of life and the conservative treatment alone seems to be ineffective in most cases. Based on the literature and personal experiences, the authors suggest 360-degree fixation of the spine to provide sufficient stability for the vertebrae of "bad bone quality", and early mobilisation of the patient can be achieved.

Screening and monitoring of the BTKC481S mutation in a real-world cohort of patients with relapsed/refractory chronic lymphocytic leukaemia during ibrutinib therapy.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has revolutionised the therapeutic landscape of chronic lymphocytic leukaemia (CLL). Acquired mutations emerging at position C481 in the BTK tyrosine kinase domain are the predominant genetic alterations associated with secondary ibrutinib resistance. To assess the correlation between disease progression, and the emergence and temporal dynamics of the most common resistance mutation BTKC481S , sensitive (10-4 ) time-resolved screening was performed in 83 relapsed/refractory CLL patients during single-agent ibrutinib treatment. With a median follow-up time of 40 months, BTKC481S was detected in 48·2% (40/83) of the patients, with 80·0% (32/40) of them showing disease progression during the examined period. In these 32 cases, representing 72·7% (32/44) of all patients experiencing relapse, emergence of the BTKC481S mutation preceded the symptoms of clinical relapse with a median of nine months. Subsequent Bcl-2 inhibition therapy applied in 28/32 patients harbouring BTKC481S and progressing on ibrutinib conferred clinical and molecular remission across the patients. Our study demonstrates the clinical value of sensitive BTKC481S monitoring with the largest longitudinally analysed real-world patient cohort reported to date and validates the feasibility of an early prediction of relapse in the majority of ibrutinib-treated relapsed/refractory CLL patients experiencing disease progression.

Involvement of the Catecholamine Pathway in Glioblastoma Development.

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). The standard of care improves the overall survival of patients only by a few months. Explorations of new therapeutic targets related to molecular properties of the tumor are under way. Even though neurotransmitters and their receptors normally function as mediators of interneuronal communication, growing data suggest that these molecules are also involved in modulating the development and growth of GBM by acting on neuronal and glioblastoma stem cells. In our previous DNA CpG methylation studies, gene ontology analyses revealed the involvement of the monoamine pathway in sequential GBM. In this follow-up study, we quantitated the expression levels of four selected catecholamine pathway markers (alpha 1D adrenergic receptor-ADRA1D; adrenergic beta receptor kinase 1 or G protein-coupled receptor kinase 2-ADRBK1/GRK2; dopamine receptor D2-DRD2; and synaptic vesicle monoamine transporter-SLC18A2) by immunohistochemistry, and compared the histological scores with the methylation levels within the promoters + genes of these markers in 21 pairs of sequential GBM and in controls. Subsequently, we also determined the promoter and gene methylation levels of the same markers in an independent database cohort of sequential GBM pairs. These analyses revealed partial inverse correlations between the catecholamine protein expression and promoter + gene methylation levels, when the tumor and control samples were compared. However, we found no differences in the promoter + gene methylation levels of these markers in either our own or in the database primary-recurrent GBM pairs, despite the higher protein expression of all markers in the primary samples. This observation suggests that regulation of catecholamine expression is only partially related to CpG methylation within the promoter + gene regions, and additional mechanisms may also influence the expression of these markers in progressive GBM. These analyses underscore the involvement of certain catecholamine pathway markers in GBM development and suggest that these molecules mediating or modulating tumor growth merit further exploration.

DNA methylation and protein expression of Wnt pathway markers in progressive glioblastoma.

BACKGROUND: Wnt signaling plays important roles in tumorigenesis, invasiveness and therapeutic resistance of glioblastoma (GBM). METHODS: We simultaneously investigated six Wnt pathway markers (Wnt5a, Fzd-2, beta-catenin, Wnt3a, Wnt7b, Fzd-10) at epigenetic and protein levels in 21 sequential formalin-fixed paraffin-embedded GBM pairs and controls. RESULTS: Expression levels of Wnt5a, beta-catenin and Wnt3a proteins either moderately or significantly increased, while those of Fzd-2, Wnt7b and Fzd-10 decreased in the primary (GBM-P) and recurrent (GBM-R) tumors compared to the controls. Methylation levels within promoters and genes showed corresponding decreases for Wnt5a, beta-catenin and Wnt3a in tumors vs. controls, while that of Fzd-10 was uniformly high. Comparing the GBM-P and GBM-R pairs, proteins of Fzd-2, beta-catenin and Wnt3a were either moderately or significantly up-, while that of Wnt7b was downregulated in GBM-R, but these patterns were not accompanied by inverse methylation patterns in the corresponding promoters and genes over time. No methylation differences were noted within promoters and genes of the same markers in 112 pairs of primary and recurrent GBMs in a database, suggesting that the observed changes in protein expression levels may not be explained by CpG methylation status alone. The promoter and gene methylation rate was the highest for Fzd-10 in the database cohort too, supporting the noted low Fzd-10 protein expression. DISCUSSION: These analyses underscore the relevance of Wnt pathway molecules in the context of their methylation profiles in the development and evolution of GBM, and suggest that Wnt pathway regulation as a potential treatment target merits further studies.

Is an Immunosuppressive Microenvironment a Characteristic of Both Intra- and Extraparenchymal Central Nervous Tumors?

In spite of intensive research, the survival rates of patients diagnosed with tumors of the central nervous system (CNS) have not improved significantly in the last decade. Immunotherapy as novel and efficacious treatment option in several other malignancies has failed in neuro-oncology likely due to the immunosuppressive property of the brain tissues. Glioblastoma (GBM) is the most aggressive malignant CNS neoplasm, while meningioma (MNG) is a mainly low grade or benign brain tumor originating from the non-glial tissues of the CNS. The aim of the current preliminary study is to compare the immune microenvironment of MNG and GBM as potential target in immunotherapy. Interestingly, the immune microenvironment of MNG and GBM have proved to be similar. In both tumors types the immune suppressive elements including regulatory T cells (Treg), tumor-associated macrophages (TAM) were highly elevated. The cytokine environment supporting Treg differentiation and the presence of indoleamine 2,3-dioxygenase 1 (IDO1) have also increased the immunosuppressive microenvironment. The results of the present study show an immune suppressive microenvironment in both brain tumor types. In a follow-up study with a larger patient cohort can provide detailed background information on the immune status of individual patients and aid selection of the best immune checkpoint inhibitor or other immune modulatory therapy. Immune modulatory treatments in combination with IDO1 inhibitors might even become alternative therapy for relapsed, multiple and/or malignant MNG or chemo-resistant GBM.

DNA CpG methylation in sequential glioblastoma specimens.

PURPOSE: Glioblastoma is the most aggressive form of brain tumors. A better understanding of the molecular mechanisms leading to its evolution is essential for the development of treatments more effective than the available modalities. Here, we aim to identify molecular drivers of glioblastoma development and recurrence by analyzing DNA CpG methylation patterns in sequential samples. METHODS: DNA was isolated from 22 pairs of primary and recurrent formalin-fixed, paraffin-embedded glioblastoma specimens, and subjected to reduced representation bisulfite sequencing. Bioinformatic analyses were conducted to identify differentially methylated sites and pathways, and biostatistics was used to test correlations among clinical and pathological parameters. RESULTS: Differentially methylated pathways likely involved in primary tumor development included those of neuronal differentiation, myelination, metabolic processes, synapse organization and endothelial cell proliferation, while pathways differentially active during glioblastoma recurrence involved those associated with cell processes and differentiation, immune response, Wnt regulation and catecholamine secretion and transport. CONCLUSION: DNA CpG methylation analyses in sequential clinical specimens revealed hypomethylation in certain pathways such as neuronal tissue development and angiogenesis likely involved in early tumor development and growth, while suggested altered regulation in catecholamine secretion and transport, Wnt expression and immune response contributing to glioblastoma recurrence. These pathways merit further investigations and may represent novel therapeutic targets.

Lipid and protein tumor markers for head and neck squamous cell carcinoma identified by imaging mass spectrometry.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To improve pre- and post-operative diagnosis and prognosis novel molecular markers are desirable. Here we used MALDI imaging mass spectrometry (IMS) and immunohistochemistry (IHC) to seek tumor specific expression of proteins and lipids in HNSCC samples. Among low molecular weight proteins visualized, S100A8 and S100A9 were found to be expressed in the regions of tumor tissue but not in the surrounding healthy stroma of a post-operative microdissected tissue. Marker potential of S100A8 and S100A9 was confirmed by immunohistochemistry of paraffin-embedded pathological samples. Imaging lipids showed a remarkable depletion of lysophosphatidylcholine species LPC[16:0], LPC[18:2] and, in parallel, accumulation of major glycerophospholipid species PE-P[36:4], PC[32:1], PC[34:1] in neoplastic areas. This was confirmed by shotgun lipidomics of dissected healthy and tumor tissue sections. A combination of the negative (LPC[16:0]) and positive (PC[32:1], PC[34:1]) markers was also applicable to uncover tumorous character of a pre-operative biopsy. Furthermore, marker potential of lysophospholipids was supported by elevated expression levels of the lysophospholipid degrading enzyme lysophospholipase A1 (LYPLA1) in the tumor regions of paraffin-embedded HNSCC samples. Finally, experimental evidence of 3D cell spheroid tests showed that LPC[16:0] facilitates HNSCC invasion, implying that HNSCC progression in vivo may be dependent on lysophospholipid supply. Altogether, a series of novel proteins and lipid species were identified by IMS and IHC screening, which may serve as potential molecular markers for tumor diagnosis, prognosis, and may pave the way to better understand HNSCC pathophyisiology.