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Objective: The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are inflammatory disorders with ANCA autoantibodies recognising either proteinase 3 (PR3-AAV) or myeloperoxidase (MPO-AAV). PR3-AAV and MPO-AAV have been associated with distinct loci in the human leucocyte antigen (HLA) region. While the association between MPO-AAV and HLA has been well characterised in East Asian populations where MPO-AAV is more common, studies in populations of European descent are limited. The aim of this study was to thoroughly characterise associations to the HLA region in Scandinavian patients with PR3-AAV as well as MPO-AAV. Methods: Genotypes of single-nucleotide polymorphisms (SNPs) located in the HLA region were extracted from a targeted exome-sequencing dataset comprising Scandinavian AAV cases and controls. Classical HLA alleles were called using xHLA. After quality control, association analyses were performed of a joint SNP/classical HLA allele dataset for cases with PR3-AAV (n=411) and MPO-AAV (n=162) versus controls (n=1595). Disease-associated genetic variants were analysed for association with organ involvement, age at diagnosis and relapse, respectively. Results: PR3-AAV was significantly associated with both HLA-DPB1*04:01 and rs1042335 at the HLADPB1 locus, also after stepwise conditional analysis. MPO-AAV was significantly associated with HLADRB1*04:04. Neither carriage of HLA-DPB1*04:01 alleles in PR3-AAV nor of HLA-DRB1*04:04 alleles in MPO-AAV were associated with organ involvement, age at diagnosis or relapse. Conclusions: The association to the HLA region was distinct in Scandinavian cases with MPO-AAV compared with cases of East Asian descent. In PR3-AAV, the two separate signals of association to the HLD-DPB1 region mediate potentially different functional effects.

Mitochondrial DNA (mtDNA) alterations have been reported in different types of cancers and are suggested to play important roles in cancer development and metastasis. However, there is little information about its involvement in pheochromocytomas and paragangliomas (PCCs/PGLs) formation. PCCs and PGLs are rare endocrine tumors of the chromaffin cells in the adrenal medulla and extra-adrenal paraganglia that can synthesize and secrete catecholamines. Over the last 3 decades, the genetic background of about 60% of PCCs/PGLs involving nuclear DNA alterations has been determined. Recently, a study showed that mitochondrial alterations can be found in around 17% of the remaining PCCs/PGLs. In this review, we summarize recent knowledge regarding both nuclear and mitochondrial alterations and their involvement in PCCs/PGLs. We also provide brief insights into the genetics and the molecular pathways associated with PCCs/PGLs and potential therapeutical targets.

Objective Autoantibodies against the adrenal enzyme 21-hydroxylase is a hallmark manifestation in autoimmune Addison's disease (AAD). Steroid 21-hydroxylase is encoded by CYP21A2, which is located in the human leucocyte antigen (HLA) region together with the highly similar pseudogene CYP21A1P. A high level of copy number variation is seen for the 2 genes, and therefore, we asked whether genetic variation of the CYP21 genes is associated with AAD. Design Case-control study on patients with AAD and healthy controls. Methods Using next-generation DNA sequencing, we estimated the copy number of CYP21A2 and CYP21A1P, together with HLA alleles, in 479 Swedish patients with AAD and autoantibodies against 21-hydroxylase and in 1393 healthy controls. Results With 95% of individuals carrying 2 functional 21-hydroxylase genes, no difference in CYP21A2 copy number was found when comparing patients and controls. In contrast, we discovered a lower copy number of the pseudogene CYP21A1P among AAD patients (P = 5 x 10(-44)), together with associations of additional nucleotide variants, in the CYP21 region. However, the strongest association was found for HLA-DQB1*02:01 (P = 9 x 10(-63)), which, in combination with the DRB1*04:04-DQB1*03:02 haplotype, imposed the greatest risk of AAD. Conclusions We identified strong associations between copy number variants in the CYP21 region and risk of AAD, although these associations most likely are due to linkage disequilibrium with disease-associated HLA class II alleles.

Pheochromocytoma and paraganglioma (PPGL), are rare neuroendocrine tumors arising from the adrenal me-dulla and extra-adrenal paraganglia, respectively. Up to about 60% are explained by germline or somatic mu-tations in one of the major known susceptibility genes e.g., in NF1, RET, VHL, SDHx, MAX and HRAS. Targeted Next Generation Sequencing was performed in 14 sporadic tumors using a panel including 26 susceptibility genes to characterize the mutation profile. A total of 6 germline and 8 somatic variants were identified. The most frequent somatic mutations were found in NF1 (36%), four have not been reported earlier in PCC or PGL. Gene expression profile analysis showed that NF1 mutated tumors are classified into RTK3 subtype, cluster 2, with a high expression of genes associated with chromaffin cell differentiation, and into a RTK2 subtype, cluster 2, as well with overexpression of genes associated with cortisol biosynthesis. On the other hand, by analyzing the entire probe set on the array and TCGA data, ALDOC was found as the most significantly down regulated gene in NF1-mutated tumors compared to NF1-wild-type. Differential gene expression analysis showed a significant difference between Nt -and Ct-NF1 domains in mutated tumors probably engaging different cellular pathways. Notably, we had a metastatic PCC with a Ct-NF1 frameshift mutation and when performing protein docking analysis, Ct-NF1 showed an interaction with Nt-FAK suggesting their involvement in cell adhesion and cell growth. These results show that depending on the location of the NF1-mutation different pathways are activated in PPGLs. Further studies are required to clarify their clinical significance., Funding Agencies|ALF Grant from Region Ostergotland; [RO-532021]

Objective: The genetic predisposition to ankylosing spondylitis (AS) has been most widely studied in cohorts with European ancestry. However, within Europe, disease prevalence is higher in Sweden. Given this, we aimed to characterize known AS susceptibility variants in a homogeneous Swedish data set, assessing reproducibility and direction of effect. Method: The power to detect association within an existing Swedish targeted sequencing study (381 controls; 310 AS cases) was examined, and a set of published associations (n = 151) was intersected with available genotypes. Association to disease was calculated using logistic regression accounting for population structure, and HLA-B27 status was determined with direct polymerase chain reaction genotyping. Results: The cases were found to be 92.3% HLA-B27 positive, with the data set showing >= 80% predictive power to replicate associations, with odds ratios >= 1.6 over a range of allele frequencies (0.1-0.7). Thirty-four markers, representing 23 gene loci, were available for investigation. The replicated variants tagged MICA and IL23R loci (p < 1.47 x 10(-3)), with variable direction of effect noted for gene loci IL1R1 and MST1. Conclusion: The Swedish data set successfully replicated both major histocompatibility complex (MHC) and non-MHC loci, and revealed a different replication pattern compared to discovery data sets. This was possibly due to population demographics, including HLA-B27 frequency and measured comorbidities., Funding Agencies|Swedish Research Council, FORMASSwedish Research CouncilSwedish Research Council Formas [Dnr 2012-1531]; Wallenberg Scholar award

Objective To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV). Methods Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay. Results PR3-ANCA+ AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 x 10-61, odds ratio (OR) 0.10; rs9277341, P = 1.5 x 10-44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 x 10-10, OR 2.9). MPO-ANCA+ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 x 10-25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 x 10-7, OR 3.0), the latter a novel susceptibility locus for MPO-ANCA+ granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele. Conclusion We identified a novel susceptibility locus for MPO-ANCA+ AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types., Funding Agencies: Swedish Society for Medical Research; Swedish Research Council; Swedish Society of Medicine; Swedish Rheumatism Association; Knut and Alice Wallenberg Foundation; AstraZeneca-Science for Life Laboratory (SciLifeLab) Research Collaboration Grant

Patients with glioblastoma (GBM) have a poor outcome, but even among patients receiving the same therapies and with good prognostic factors, one can find those with exceptionally short and long survival. From the Nordic trial, which randomized GBM patients of 60 years or older between two radiotherapy arms (60 Gy or 34 Gy) or temozolomide (TMZ), we selected 59 with good prognostic factors. These selected GBM patients were equally distributed according to treatment and MGMT promoter methylation status but had long or short survival. Methylation profiling with the Illumina Infinium Methylation EPIC BeadChip arrays was performed and utilized for methylation-based CNS tumor classification, and pathway enrichment analysis of differentially methylated CpG sites (DMCs), as well as calculation of epigenetic age acceleration with three different algorithms, to compare the long and short survival groups. Samples identified by the classifier as non-GBM IDH wildtype were excluded. DMCs between long- and short-term survivors were found in patients with methylated MGMT promoter treated with TMZ (123,510), those with unmethylated MGMT treated with 60Gy radiotherapy (4,086), and with methylated MGMT promoter treated with 34Gy radiotherapy (39,649). Long-term survivors with methylated MGMT promoter treated with TMZ exhibited hypermethylation of the Wnt signaling and the platelet activation, signaling, and aggregation pathways. The joint analysis of radiotherapy arms revealed 319 DMCs between long- and short-term survivors with unmethylated MGMT and none for samples with methylated MGMT promoter. An analysis comparing epigenetic age acceleration between patients with long- and short-term survival across all treatment arms showed a decreased epigenetic age acceleration for the latter. We identified DMCs for both TMZ and RT-treated patients and epigenetic age acceleration as a potential prognostic marker, but further systematic analysis of larger patient cohorts is necessary for confir, Funding Agencies|Swedish Cancer Foundation; Lions Research Fund; FORSS; LiU Cancer

Background: Enzymes of tricarboxylic acid (TCA) have recently been recognized as tumor suppressors. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) cause pheochromocytomas and paragangliomas (PCCs/PGLs) and predispose patients to malignant disease with poor prognosis. Methods: Using the human pheochromocytoma cell line (hPheo1), we knocked down SDHB gene expression using CRISPR-cas9 technology. Results: Microarray gene expression analysis showed that >500 differentially expressed gene targets, about 54%, were upregulated in response to SDHB knock down. Notably, genes involved in glycolysis, hypoxia, cell proliferation, and cell differentiation were up regulated, whereas genes involved in oxidative phosphorylation (OXPHOS) were downregulated. In vitro studies show that hPheo1 proliferation is not affected negatively and the cells that survive by shifting their metabolism to the use of glutamine as an alternative energy source and promote OXPHOS activity. Knock down of SDHB expression results in a significant increase in GLUD1 expression in hPheo1 cells cultured as monolayer or as 3D culture. Analysis of TCGA data confirms the enhancement of GLUD1 in SDHB mutated/low expressed PCCs/PGLs. Conclusions: Our data suggest that the downregulation of SDHB in PCCs/PGLs results in increased GLUD1 expression and may represent a potential biomarker and therapeutic target in SDHB mutated tumors and SDHB loss of activity-dependent diseases., Funding: This study has been supported by the ALF Grant from Region Östergötland (RÖ-6079119), a grant from the Lisa och Johan Grönbergs Foundation (2019-00175) and by a grant from LiU Cancer, Linköping University (LiU-2017; LiU-2019).

INTRODUCTION: Genetic variants in the Beta-glucocerebrosidase gene (GBA1) is a known risk factor for Parkinson's disease. The GBA1 mutations L444P, N370S and many other have been shown to associate with the disease in populations with diverse background. Some GBA1 polymorphisms have a less pronounced effect, and their pathogenicity has been debated. We have previously found associations with L444P, N370S and E326K and Parkinson's disease in Sweden. METHOD: In this study we used pyrosequencing to genotype the T369M variant in a large Swedish cohort consisting of 1,131 patients with idiopathic Parkinson's disease, and 1,594 control subjects to evaluate the possibility of this variant conferring an increased risk for Parkinson's disease. RESULTS: The minor allele frequency was 2.15% in patients and 1.76% in controls. Statistical analysis showed that there was no significant difference in allele frequency between patients and control subjects, p-value 0.37, Odds Ratio 1.23 with a 95% confidence interval of 0.82-1.83. CONCLUSION: Our results suggest that T369M is not a risk factor for Parkinson's disease in the Swedish population., Funding agencies: Swedish Research Council, The Swedish Parkinson Foundation, The Swedish Brain Foundation, Swedish Brain Power and the Karolinska Institutet Foundation and Funds, PS is a Wallenberg Clinical Scholar

Genetic testing has become the standard of care for many disease states. As a result, physicians treating patients who have tumors often rely on germline genetic testing results for making clinical decisions. Cases of two sisters carrying a germline CHEK2 variant are highlighted whereby possible other genetic drivers were discovered on tumor analysis. CHEK2 (also referred to as CHK2) loss of function has been firmly associated with breast cancer development. In this case report, two siblings with a germline CHEK2 mutation also had distinct endocrine tumors. Pituitary adenoma and pancreatic neuroendocrine tumor (PNET) was found in the first sibling and pheochromocytoma (PCC) discovered in the second sibling. Although pituitary adenomas, PNETs, and PCC have been associated with NF1 gene mutations, the second sister with a PCC did have proven germline CHEK2 with a pathogenic somatic NF1 mutation. We highlight the clinical point that unless the tumor is sequenced, the real driver mutation that is causing the patients tumor may remain unknown., Funding Agencies|NIGMS [GM114102]; CTSA-IIMS (NIH/NCATS Grant) [UL1TR001120, UL1 TR002645]; Mays Cancer Center NIH-NCI [P30 CA54174]; Alexs Lemonade Stand Foundation; Northwest Mutual/Flashes of Hope; Gatorade Trust

Objective To study the telomerase reverse transcriptase (TERT) mutation and the p53 pathway in T1 urinary bladder cancer (UBC). Materials and Methods This prospectively performed population-based study included all patients in the Southeast Healthcare Region in Sweden with T1 UBC registered in the period 1992-2001, inclusive. Given that p53 and TERT are important factors for tumour proliferation, although their interrelationships are unknown, we assessed both the TERT and the p53 mutations. Furthermore, we conducted a p53 immunohistochemistry (IHC) analysis using two thresholds for p53 positivity: 10% of tumour cells and 50% of tumour cells (p53 IHC50%). Cox proportional hazards analysis and Kaplan-Meier curves were used to study time to tumour progression. Results Out of 158 patients, we observed the TERT mutation in 74 (47%), the p53 mutation in 48 (30%), and p53 IHC50% positivity in 72 patients (46%). The TERT mutation was more common in p53 mutation-positive patients (P = 0.009), and the latter group also had more patients with p53 IHC50%-positive tumour cells (P = 0.02). In the TERT mutation-negative tumours a p53-positive mutation was associated with a shorter time to progression (P = 0.03) compared to patients with p53-negative mutation. In contrast, in tumours with both TERT mutation positivity and p53 mutation positivity, a longer time to progression was observed in the group with p53 IHC50% positivity compared to the group with p53 IHC50%-negative tumours. Conclusions In stage T1 UBC, the combination of the TERT mutation and the p53 mutation was associated with tumour progression. A protective effect of the TERT promotor mutation against tumour progression induced by the p53 mutation and subsequent p53 accumulation in tumour cells might be possible, but further investigations are necessary.

Background Autosomal recessive congenital hereditary corneal dystrophy (CHED) is a rare isolated developmental anomaly of the eye characterised by diffuse bilateral corneal clouding that may lead to visual impairment requiring corneal transplantation. CHED is known to be caused by mutations in the solute carrier family 4 member 11 (SLC4A11) gene which encodes a membrane transporter protein (sodium bicarbonate transporter-like solute carrier family 4 member 11). Methods To identify SLC4A11 gene mutations associated with CHED (OMIM: #217700), genomic DNA was extracted from whole blood and sequenced for all exons and intron-exon boundaries in two large Tunisian families. Results A novel deletion SLC4A11 mutation (p. Leu479del; c.1434_1436del) is responsible for CHED in both analysed families. This non-frameshift mutation was found in a homozygous state in affected members and heterozygous in non-affected members. In silico analysis largely support the pathogenicity of this alteration that may leads to stromal oedema by disrupting the osmolarity balance. Being localised to a region of alpha-helical secondary structure, Leu479 deletion may induce protein-compromising structural rearrangements. Conclusion To the best of our knowledge, this is the first clinical and genetic study exploring CHED in Tunisia. The present work also expands the list of pathogenic genotypes in SLC4A11 gene and its associated clinical diagnosis giving more insights into genotype-phenotype correlations., Funding Agencies|Ministry of Higher Education and Scientific Research-Tunisia [LR16E16]; Foundation of Synframjandets Forskningsfond/Ogonfonden

Objective: Psoriatic arthritis (PsA) is a heterogeneous inflammatory disease associated with psoriasis. Underlying genetic factors are considered important for disease expression and prognosis of PsA. Interleukin-1β-regulating protein complexes called inflammasomes are associated with several inflammatory diseases, e.g. rheumatoid arthritis and psoriasis. The aim was to determine whether inflammasome-related genetic variation is associated with PsA susceptibility or different disease phenotypes. Method: DNA from 724 patients with PsA and 587 population-based controls from northern Sweden was analysed for single-nucleotide polymorphisms in NLRP3-Q750K (rs35829419), NLRP3 (rs10733113), CARD8-C10X (rs2043211), NLRP1 (rs8079034), and NLRP1 (rs878329). Results: Significant associations were found with the genotype AA (vs AT+TT) of rs2043211 for PsA patients compared with controls [odds ratio (OR), 95% confidence interval (CI) 1.32 (1.05–1.65), p = 0.016]; and between the C-allele of rs878329 and axial involvement of PsA [OR (95% CI) 1.37 (1.02–1.84), p = 0.035], the T-allele of rs8079034 with prescription of conventional synthetic disease-modifying anti-rheumatic drugs [OR (95% CI) 1.76 (1.23–2.53), p = 0.0020], the G-allele of rs10733113 and patients with a skin disease with early onset [OR (95% CI) 1.58 (1.13–2.21), p = 0.007], and the C-allele of rs35829419 and a destructive/deforming disease [OR (95% CI) 1.63 (1.04–2.55), p = 0.030]. Conclusions: This study is the first to show an association with a genetic polymorphism in an inflammasome-related gene, CARD8-C10X (rs2043211), in patients with PsA. Associations between different phenotypes of PsA and different polymorphisms of the inflammasome genes were also found. Our results indicate the involvement of inflammasome genes in the pathogenesis and disease expression of PsA., Funding Agencies|Psoriasisforbundet

Objectives: To further elucidate the role of the MHC in ankylosing spondylitis by typing 17 genes, searching for HLA-B∗27 independent associations and assessing the impact of sex on this male biased disease. Methods: High-confidence two-field resolution genotyping was performed on 310 cases and 2196 controls using an n-1 concordance method. Protein-coding variants were called from next-generation sequencing reads using up to four software programs and the consensus result recorded. Logistic regression tests were applied to the dataset as a whole, and also in stratified sets based on sex or HLA-B∗27 status. The amino acids driving association were also examined. Results: Twenty-five HLA protein-coding variants were significantly associated to disease in the population. Three novel protective associations were found in a HLA-B∗27 positive population, HLA-A∗24:02 (OR = 0.4, CI = 0.2–0.7), and HLA-A amino acids Leu95 and Gln156. We identified a key set of seven loci that were common to both sexes, and robust to change in sample size. Stratifying by sex uncovered three novel risk variants restricted to the female population (HLA-DQA1∗04.01, -DQB1∗04:02, -DRB1∗08:01; OR = 2.4–3.1). We also uncovered a set of neutral variants in the female population, which in turn conferred strong effects in the male set, highlighting how population composition can lead to the masking of true associations. Conclusion: Population stratification allowed for a nuanced investigation into the tightly linked MHC region, revealing novel HLA-B∗27 signals as well as replicating previous HLA-B∗27 dependent results. This dissection of signals may help to elucidate sex biased disease predisposition and clinical progression., Funding agencies: Swedish Research Council, FORMAS (Dnr 2012-1531), Knut and Alice Wallenberg Foundation (Dnr 2012-0268), Swedish Research Council for Medicine and Health (D0283001 and Dnr 2018-02399), the Swedish Rheumatism Association, and King Gustaf V’s 80-year Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer and its incidence is increasing. Preoperative diagnosis is warranted in order to avoid two-stage procedures that are associated with additional costs and higher radioactive iodine remnant uptake. In the setting of thyroid cancer, somatic BRAF V600E-mutations are highly specific for PTC and can be analyzed in aspirates from fine-needle aspiration cytology (FNAC). The gold standard to perform FNAC is ultrasound guidance. Here, we analyze whether adding BRAF V600E-mutation analysis could be of value in palpation-guided FNACs. A total of 430 consecutive patients were included. Ultrasound-guided FNACs were performed in 251 patients and 179 patients underwent palpation-guided FNACs. BRAF V600E-mutation analysis was performed using two methods, an allele-specific polymerase chain reaction (PCR) analyzed by capillary gel electrophoresis (PCR/Qiaxcel), and a droplet digital PCR (ddPCR) assay. A total of 80 patients underwent surgery, and histology revealed 25 patients to have PTC. Of the 25 PTCs, 23 (92%) showed a BRAF V600E-mutation. Both mutation analysis methods (PCR/Qiaxcel and ddPCR) produced concordant results. In the ultrasound-guided group, the preoperative diagnostic sensitivity of FNAC using the Bethesda classification alone was very high and additional BRAF V600E-mutation analysis added little to the preoperative diagnostic sensitivity. By contrast, in the palpation-guided group, by adding BRAF V600E-mutation analysis, eight instead of four patients were diagnosed of having PTC. This increase in the diagnostic sensitivity was statistically significant (p < 0.05). The costs per sample were as low as 62 USD (PCR/Qiaxcel and ddPCR) and 35 USD (PCR/Qiaxcel only). Ultrasound-guided FNAC should be aimed for when dealing with thyroid nodules. However, if palpation-guided FNAC cannot be avoided or may be required due to resource utilization, adding BRAF V600E-mutation analysis using the met, Funding Agencies|Medical Research Council of Southeast SwedenUK Research & Innovation (UKRI)Medical Research Council UK (MRC) [FORSS-648671, FORSS-861891]

Simple Summary Glioblastoma (GBM) is one of the most common and most aggressive brain tumors with higher prevalence among men than women. Loss of chromosome Y (LOY) in the peripheral blood cells has been associated with increased risk of developing cancer. However, there is a lack of data about LOY in GBM tumor tissue and the potential impact on patients prognosis. Through droplet digital PCR (ddPCR) analysis of 10 markers spread throughout chromosome Y in 105 male GBM patients, we were able to identify deletion of SRY gene as a factor strongly correlating with reduced overall survival. This finding was later corroborated by the analysis of GBM gene expression data collected in TCGA, showing correlation between decreased SRY expression and shortened overall survival. Background: Biological causes of sex disparity seen in the prevalence of cancer, including glioblastoma (GBM), remain poorly understood. One of the considered aspects is the involvement of the sex chromosomes, especially loss of chromosome Y (LOY). Methods: Tumors from 105 isocitrate dehydrogenase (IDH) wild type male GBM patients were tested with droplet digital PCR for copy number changes of ten genes on chromosome Y. Decreased gene expression, a proxy of gene loss, was then analyzed in 225 IDH wild type GBM derived from TCGA and overall survival in both cohorts was tested with Kaplan-Meier log-rank analysis and maximally selected rank statistics for cut-off determination. Results: LOY was associated with significantly shorter overall survival (7 vs. 14.6 months, p = 0.0016), and among investigated individual genes survival correlated most prominently with loss of the sex-determining region Y gene (SRY) (10.8 vs. 14.8 months, p = 0.0031). Gene set enrichment analysis revealed that epidermal growth factor receptor, platelet-derived growth factor receptor, and MYC proto-oncogene signaling pathways are associated with low SRY expression. Conclusion: Our data show that deletions and reduced gene expressio, Funding Agencies|Erik, Karin and Gosta Selanders Foundation at Uppsala University Hospital; Swedish Cancer Foundation; ALF Grants; Region Ostergotland; Medical Research Council of Southeast SwedenUK Research & Innovation (UKRI)Medical Research Council UK (MRC); LiU Cancer at Linkoping University; Lions Cancer Foundation

INTRODUCTION: Recent studies suggest an overrepresentation of MGMT promoter methylated tumors in females with IDHwt glioblastoma (GBM) compared to males, with a subsequent better response to alkylating treatment. METHODS: To reveal sex-bound associations that may have gone unnoticed in the original analysis, we re-analyzed two previously published clinical cohorts. One was the multicenter Nordic trial of elderly patients with GBM, randomizing patients into three different treatment arms, including 203 cases with known MGMT promoter methylation status. The other was a population-based study of 179 patients with IDHwt GBM, receiving concomittant radiotherapy and chemotherapy with temozolomide. Cohorts were stratified by sex to test the hypothesis that female sex in combination with MGMT promoter methylation constitutes a subgroup with more favorable outcome. RESULTS: There was a significantly larger proportion of MGMT promoter methylation and better outcome for female patients with MGMT promoter methylated tumors. Results were confirmed in 257 TCGA-derived IDHwt GBM with known sex and MGMT status. CONCLUSIONS: These results confirm that patient sex in combination with MGMT promoter methylation is a key determinant in GBM to be considered prior to treatment decisions. Our study also illustrates the need for stratification to identify such sex-bound associations.

Background: Glioma O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status informs clinical decision making. Worldwide different methods and cutoff levels are used, which can lead to discordant methylation results. Methods: We conducted an international survey to clarify which methods are regularly used and why. We also explored opinions regarding international consensus on methods and cutoff. Results: The survey had 152 respondents from 25 countries. MGMT methylation status is determined for all glioblastomas in 37% of laboratories. The most common methods are methylation-specific polymerase chain reaction (msPCR) (37%) and pyrosequencing (34%). A method is selected for simplicity (56%), cost-effectiveness (50%), and reproducibility of results (52%). For sequencing, the number of CpG sites analyzed varies from 1–3 up to more than 16. For 50% of laboratories, the company producing the kit determines which CpG sites are examined, whereas 33% select the sites themselves. Selection of cutoff is equally distributed among a cutoff defined in the literature, by the local laboratory, or by the outside laboratory performing the analysis. This cutoff varies, reported from 1% to 30%, and in 1 laboratory tumor is determined as methylated in case of 1 methylated CpG site of 17 analyzed. Some report tumors as unmethylated or weakly vs highly methylated. An international consensus on MGMT methylation method and cutoff is warranted by 66% and 76% of respondents, respectively. The method preferred would be msPCR (45%) or pyrosequencing (42%), whereas 18% suggest next-generation sequencing. Conclusion: Although analysis of MGMT methylation status is routine, there is controversy regarding laboratory methods and cutoff level. Most respondents favor development of international consensus guidelines.

Phaeochromocytomas and paragangliomas (PPGLs) are neuroendocrine catecholamine-producing tumours that may progress into inoperable metastatic disease. Treatment options for metastatic disease are limited, indicating a need for functional studies to identify pharmacologically targetable pathophysiological mechanisms, which require biologically relevant experimental models. Recently, a human progenitor phaeochromocytoma cell line named "hPheo1" was established, but its genotype has not been characterised. Performing exome sequencing analysis, we identified a KIF1B T827I mutation, and the oncogenic NRAS Q61K mutation. While KIF1B mutations are recurring somatic events in PPGLs, NRAS mutations have hitherto not been detected in PPGLs. Therefore, we aimed to assess its implications for the hPheo1 cell line, and possible relevance for the pathophysiology of PPGLs. We found that transient downregulation of NRAS in hPheo1 led to elevated expression of genes associated with cell adhesion, and enhanced adhesion to hPheo1 cells extracellular matrix. Analyses of previously published mRNA data from two independent PPGL patient cohorts (212 tissue samples) revealed a subcluster of PPGLs featuring hyperactivated RAS pathway-signalling and under-expression of cell adhesion-related gene expression programs. Thus, we conclude that NRAS activity in hPheo1 decreases adhesion to their own extracellular matrix and mirrors a transcriptomic RAS-signalling-related phenomenon in PPGLs., Funding Agencies|ALF Grants, Region Ostergotland [LIO-532021]; Medical Research Council of Southeast Sweden [FORSS-481781]; LiU Cancer, Linkoping University