Somatic RIT1 delins in arteriovenous malformations hyperactivate RAS-MAPK signaling amenable to MEK inhibition.

Arteriovenous malformations (AVM) are benign vascular anomalies prone to pain, bleeding, and progressive growth. AVM are mainly caused by mosaic pathogenic variants of the RAS-MAPK pathway. However, a causative variant is not identified in all patients. Using ultra-deep sequencing, we identified novel somatic RIT1 delins variants in lesional tissue of three AVM patients. RIT1 encodes a RAS-like protein that can modulate RAS-MAPK signaling. We expressed RIT1 variants in HEK293T cells, which led to a strong increase in ERK1/2 phosphorylation. Endothelial-specific mosaic overexpression of RIT1 delins in zebrafish embryos induced AVM formation, highlighting their functional importance in vascular development. Both ERK1/2 hyperactivation in vitro and AVM formation in vivo could be suppressed by pharmacological MEK inhibition. Treatment with the MEK inhibitor trametinib led to a significant decrease in bleeding episodes and AVM size in one patient. Our findings implicate RIT1 in AVM formation and provide a rationale for clinical trials with targeted treatments.

Non-Hotspot PIK3CA Variants Have Higher Variant Allele Frequency and are More Common in Syndromic Vascular Malformations.

PIK3CA variants are known to cause vascular malformations. We were interested in studying the phenotypic spectrum, the location within the PIK3CA gene, and the variant allele frequency (VAF) of somatic PI3KCA variants in vascular malformations. Clinical data of consecutive patients with extracranial/extraspinal vascular malformations were collected in the context of the VASCOM cohort (2008-2022, n = 558). Starting October 2020, biopsy samples were tested with the TSO500 gene panel (Illumina). All consenting patients with PIK3CA variants were included in this study. Eighty-nine patients had available genetic results by June 2022. PIK3CA variants (n = 25) were found in 16 simple/combined (nonsyndromic) vascular malformations and in nine vascular malformations associated with other anomalies (syndromic). Four hotspot variants in exons 9 and 20 (c.1624G>A, c.1633G>A, c.3140A>G, c.3140A>T) were identified in 16/25 patients (VAF 0.9%-9.7%). Six non-hotspot variants (c.328_330del, c.323_337del, c.353G>A, c.1258T>C, c.3132T>A, c.3195_3203delinsT) were detected in nine patients (VAF 3.6%-31.7%). Non-hotspot variants were more frequent in syndromic than nonsyndromic vascular malformations (p = 0.0034) and exhibited a higher VAF than hotspot variants (p = 0.0253). Our study contributes to the growing body of knowledge of the genetic background in vascular malformations. Further studies will enrich the ever-growing list of pathogenic PIK3CA variants associated with vascular malformations.

Tumor Necrosis Factor-α Initiates miRNA-mRNA Signaling Cascades in Obstruction-Induced Bladder Dysfunction.

Bladder outlet obstruction (BOO) and the ensuing clinical lower urinary tract dysfunction are common in elderly patients. BOO is accompanied by urodynamic changes in bladder function and leads to organ fibrosis and ultimately loss of contractility. Comprehensive transcriptome analysis of bladder samples from human patients with different urodynamically defined phenotypes of BOO revealed tumor necrosis factor (TNF)-α as the top upstream signaling pathway regulator. Herein, we validated next-generation sequencing and pathway analysis in cell-based models using bladder smooth muscle and urothelial cells exposed to TNF-α. miRNA profiling and transcriptome analysis of TNF-α-treated bladder smooth muscle cells revealed striking similarities with human BOO. Using a comparative approach, TNF-specific and TNF-independent pathways were delineated in human biopsy specimens. Concomitant down-regulation of smooth muscle cell-specific miRNAs and smooth muscle markers after TNF-α treatment was in accordance with the loss of contractility in humans in advanced obstruction-induced bladder remodeling. The expression levels of four abundant TNF-regulated miRNAs were modulated; the compensatory up-regulation of miR-199a-5p reduced NF-κB signaling. Essential hubs of TNF-α signaling pathways mitogen-activated protein kinase kinase kinase (apoptosis signal-regulating kinase 1) and inhibitor of nuclear factor κ B kinase subunit ß (IκB kinase ß) were targeted by miR-199a-5p. miR-199a-5p might be part of a negative feedback loop, reducing the impact of TNF, whereas its down-regulation in acontractile bladders from BOO patients advances the disease. The compensatory up-regulation of miR-199a-5p together with TNF-α inhibition may be therapeutically beneficial.

miR-19b enhances proliferation and apoptosis resistance via the EGFR signaling pathway by targeting PP2A and BIM in non-small cell lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) mutations enable constitutive active downstream signaling of PI3K/AKT, KRAS/ERK and JAK/STAT pathways, and promote tumor progression by inducing uncontrolled proliferation, evasion of apoptosis and migration of non-small cell lung cancer (NSCLC). In addition, such EGFR mutations increase the susceptibility of patients with NSCLC to tyrosine kinase inhibitor (TKI) therapy, but treated patients will invariably relapse with resistant disease. A global understanding of underlying molecular mechanisms of EGFR signaling may improve the management of NSCLC patients. METHODS: microarray analysis was performed to identify PI3K/AKT-regulated miRNAs. Phosphoproteomic analysis and cell based assays were performed using NSCLC cell lines lentivirally transduced with anti-miR or miR overexpressing constructs. RESULTS: Here, we show that 17 miRNAs including members of the miR-17~ 92 cluster are dysregulated following PI3K/AKT inhibition of EGFR mutant NSCLC cells. Bioinformatics analysis revealed that dysregulated miRNAs act in a concerted manner to enhance the activity of the EGFR signaling pathway. These findings were closely mirrored by attenuation of miR-17~ 92 family member miR-19b in NSCLC cell lines which resulted in reduced phosphorylation of ERK, AKT and STAT and effector proteins in EGFR mutant NSCLC cells. Consistent with this finding, cell cycle progression, clonogenic growth and migration were reduced and apoptosis was enhanced. Co-treatment of NSCLC cells with the tyrosine kinase inhibitor (TKI) gefitinib and anti-miR-19b construct reduced migration and clonogenic growth in a synergistic manner suggesting that EGFR and miR-19b act together to control oncogenic processes. Serine/threonine phosphatase PP2A subunit PPP2R5E and BCL2L11 encoding BIM were identified as major targets of miR-19b by target validation assays. Consistent with this finding, PP2A activity was strongly enhanced in NSCLC transduced with anti-miR-19b construct, but not in cells co-transduced with anti-miR-19b and shPPP2R5E, suggesting that PPP2R5E is a major constituent of the PP2A complex. Accordingly, enhanced proliferation by miR-19b was due to targeting PPP2R5E. In contrast, apoptosis resistance was mainly due to targeting BCL2L11. CONCLUSION: Our results provide insight into the importance of targeting PPP2R5E and BCL2L11 by miR-19b in oncogenic processes of NSCLC. Attenuation of miR-19b expression could potentially be exploited in adjuvant therapy of EGFR mutant NSCLC.

MicroRNA dysregulation in the tumor microenvironment influences the phenotype of pancreatic cancer.

Cellular interactions in the tumor microenvironment influence neoplastic progression in pancreatic ductal adenocarcinoma. One underlying mechanism is the induction of the prognostically unfavorable epithelial-mesenchymal-transition-like tumor budding. Our aim is to explore the expression of microRNAs implicated in the regulation of tumor budding focusing on the microenvironment of the invasive front. To this end, RNA from laser-capture-microdissected material of the main tumor, tumor buds, juxta-tumoral stroma, tumor-remote stroma, and non-neoplastic pancreatic parenchyma from pancreatic cancer cases with (n=7) and without (n=6) tumor budding was analyzed by qRT-PCR for the expression of a panel of miRNAs that are known to be implicated in the regulation of epithelial-mesenchymal transition, including miR-21, miR-183, miR-200b, miR-200c, miR-203, miR-205, miR-210, and miR-217. Here we show that at the invasive front of pancreatic ductal adenocarcinoma, specific microRNAs, are differentially expressed between tumor buds and main tumor cells and between cases with and without tumor budding, indicating their involvement in the regulation of the budding phenotype. Notably, miR-200b and miR-200c were significantly downregulated in the tumor buds. Consistent with this finding, they negatively correlated with the expression of epithelial-mesenchymal-transition-associated E-cadherin repressors ZEB1 and ZEB2 in the budding cells (P<0.001). Interestingly, many microRNAs were also dysregulated in juxta-tumoral compared to tumor-remote stroma suggesting that juxta-tumoral stroma contributes to microRNA dysregulation. Notably, miR-200b and miR-200c were strongly downregulated while miR-210 and miR-21 were upregulated in the juxta-tumoral vs tumor-remote stroma in carcinomas with tumor budding. In conclusion, microRNA targeting in both tumor and stromal cells could represent a treatment option for aggressive pancreatic cancer.

Combined ATRX/IDH1 immunohistochemistry predicts genotype of oligoastrocytomas.

AIMS: To assess whether in oligoastrocytomas ATRX deficiency, as a surrogate of the alternative lengthening of telomeres (ALT) pathway, has a role in predicting the presence or absence of loss of heterozygosity (LOH) of 1p and 19q, the genetic signature of oligodendroglial differentiation and a favourable prognostic marker. METHODS AND RESULTS: A series of 54 oligoastrocytomas were investigated by immunohistochemistry as well as microsatellite analysis for LOH 1p19q. Genetic findings were correlated with morphological assessment. CONCLUSIONS: ATRX deficiency was mutually exclusive with LOH. Conversely, ATRX-proficient tumours immunoreactive for R132H-mutant isocitrate dehydrogenase 1 (IDH1) showed a high rate (85%) of LOH. A more oligodendroglioma-like morphology was associated with a higher rate of LOH even in the morphologically ambiguous group of oligoastrocytomas. Our findings support the concept that oligoastrocytomas represent a morphological grey zone, rather than a group of truly 'mixed' or 'intermediate' tumours. More precise classification of diffuse gliomas may also improve grading of borderline cases. We propose an immunohistochemical algorithm for classification of morphologically ambiguous diffuse gliomas.

Loss of DAXX and ATRX are associated with chromosome instability and reduced survival of patients with pancreatic neuroendocrine tumors.

BACKGROUND & AIMS: Sporadic pancreatic neuroendocrine tumors (pNETs) are rare and genetically heterogeneous. Chromosome instability (CIN) has been detected in pNETs from patients with poor outcomes, but no specific genetic factors have been associated with CIN. Mutations in death domain-associated protein gene (DAXX) or ATR-X gene (ATRX) (which both encode proteins involved in chromatin remodeling) have been detected in 40% of pNETs, in association with activation of alternative lengthening of telomeres. We investigated whether loss of DAXX or ATRX, and consequent alternative lengthening of telomeres, are related to CIN in pNETs. We also assessed whether loss of DAXX or ATRX is associated with specific phenotypes of pNETs. METHODS: We collected well-differentiated primary pNET samples from 142 patients at the University Hospital Zurich and from 101 patients at the University Hospital Bern (both located in Switzerland). Clinical follow-up data were obtained for 149 patients from general practitioners and tumor registries. The tumors were reclassified into 3 groups according to the 2010 World Health Organization classification. Samples were analyzed by immunohistochemistry and telomeric fluorescence in situ hybridization. We correlated loss of DAXX, or ATRX, expression, and activation of alternative lengthening of telomeres with data from comparative genomic hybridization array studies, as well as with clinical and pathological features of the tumors and relapse and survival data. RESULTS: Loss of DAXX or ATRX protein and alternative lengthening of telomeres were associated with CIN in pNETs. Furthermore, loss of DAXX or ATRX correlated with tumor stage and metastasis, reduced time of relapse-free survival, and decreased time of tumor-associated survival. CONCLUSIONS: Loss of DAXX or ATRX is associated with CIN in pNETs and shorter survival times of patients. These results support the hypothesis that DAXX- and ATRX-negative tumors are a more aggressive subtype of pNET, and could lead to identification of strategies to target CIN in pancreatic tumors.

Prognostic and predictive roles of MGMT protein expression and promoter methylation in sporadic pancreatic neuroendocrine neoplasms.

BACKGROUND/AIMS: O(6)-methylguanine-methyltransferase (MGMT) is an important enzyme of DNA repair. MGMT promoter methylation is detectable in a subset of pancreatic neuroendocrine neoplasms (pNEN). A subset of pNEN responds to the alkylating agent temozolomide (TMZ). We wanted to correlate MGMT promoter methylation with MGMT protein loss in pNEN, correlate the findings with clinico-pathological data and determine the role of MGMT to predict response to TMZ chemotherapy. METHODS: We analysed a well-characterized collective of 141 resected pNEN with median follow-up of 83 months for MGMT protein expression and promoter methylation using methylation-specific PCR (MSP). A second collective of 10 metastasized, pretreated and progressive patients receiving TMZ was used to examine the predictive role of MGMT by determining protein expression and promoter methylation using primer extension-based quantitative PCR. RESULTS: In both collectives there was no correlation between MGMT protein expression and promoter methylation. Loss of MGMT protein was associated with an adverse outcome, this prognostic value, however, was not independent from grade and stage in multivariate analysis. Promoter hypermethylation was significantly associated with response to TMZ. CONCLUSION: Loss of MGMT protein expression is associated with adverse outcome in a surgical series of pNET. MGMT promoter methylation could be a predictive marker for TMZ chemotherapy in pNEN, but further, favourably prospective studies will be needed to confirm this result and before this observation can influence clinical routine.

Anaplastic oligodendroglioma arising from the brain stem and featuring 1p/19q co-deletion.

With respect to localization, oligodendrogliomas are characterized by a marked preponderance of the cerebral hemispheres. Outside these typical sites, any tumor histopathologically reminiscent of oligodendroglioma a priori is likely to represent one of its morphological mimics, including clear cell ependymoma, neurocytoma, pilocytic astrocytoma or glioneuronal tumors. This is particularly relevant as several of the latter are in principle curable by surgery. Among extrahemispherical sites, bona fide oligodendroglioma - as characterized by loss of heterozygosity (LOH) of chromosome arms 1p and 19q - so far has not been documented to occur in the brain stem. Here, we report the case of a 55-year-old female patient with an anaplastic oligodendroglioma (WHO grade III) of the brain stem and cerebellum diagnosed by stereotactic biopsy and featuring combined LOH of 1p and 19q. A morphological peculiarity was a population of interspersed tumor giant cells, a phenomenon that has been referred to as polymorphous oligodendroglioma. Our findings confirm the notion that - although very infrequently - true oligodendrogliomas do occur in the infratentorial compartment.

Polymorphous oligodendroglioma of Zülch revisited: a genetically heterogeneous group of anaplastic gliomas including tumors of bona fide oligodendroglial differentiation.

A polymorphous variant of oligodendroglioma was described by K.J. Zülch half a century ago, and is only very sporadically referred to in the subsequent literature. In particular, no comprehensive analysis with respect to clinical or genetic features of these tumors is available. From a current perspective, the term polymorphous oligodendroglioma (pO) may appear as contradictory in terms, as nuclear monotony is a histomorphological hallmark of oligodendrogliomas. For the purpose of this study, we defined pO as diffusely infiltrating gliomas felt to be of oligodendroglial rather than astrocytic differentiation and characterized by the presence of multinucleate tumor giant cells and/or nuclear pleomorphism. In a total of nine patients, we identified tumors consistent with this working definition. All tumors were high-grade. We characterized these with respect to clinical, histomorphological and genetic features. Despite clinical and genetic heterogeneity, we identified a subset of tumors of bona fide oligodendroglial differentiation as characterized by combined loss of heterozygosity of chromosome arms 1p and 19q (LOH 1p19q). Those tumors that lacked LOH 1p19q showed a high frequency of IDH1 mutations and loss of alpha thalassemia/mental retardation syndrome X-linked gene (ATRX) immunoreactivity, indicating a possible phenotypic convergence of true oligodendrogliomas and gliomas of the alternative lengthening of telomeres (ALT) pathway. p53 alterations were common irrespective of the 1p19q status. Histomorphologically, the tumors featured interspersed bizarre multinucleate giant tumor cells, while the background population varied from monotonous to significantly pleomorphic. Our findings indicate, that a rare polymorphous - or "giant cell" - variant of oligodendroglioma does indeed exist.

Methods in cancer research: Assessing therapy response of spheroid cultures by life cell imaging using a cost-effective live-dead staining protocol.

Spheroid cultures of cancer cell lines or primary cells represent a more clinically relevant model for predicting therapy response compared to two-dimensional cell culture. However, current live-dead staining protocols used for treatment response in spheroid cultures are often expensive, toxic to the cells, or limited in their ability to monitor therapy response over an extended period due to reduced stability. In our study, we have developed a cost-effective method utilizing calcein-AM and Helix NP™ Blue for live-dead staining, enabling the monitoring of therapy response of spheroid cultures for up to 10 days. Additionally, we used ICY BioImage Analysis and Z-stacks projection to calculate viability, which is a more accurate method for assessing treatment response compared to traditional methods on spheroid size. Using the example of glioblastoma cell lines and primary glioblastoma cells, we show that spheroid cultures typically exhibit a green outer layer of viable cells, a turquoise mantle of hypoxic quiescent cells, and a blue core of necrotic cells when visualized using confocal microscopy. Upon treatment of spheroids with the alkylating agent temozolomide, we observed a reduction in the viability of glioblastoma cells after an incubation period of 7 days. This method can also be adapted for monitoring therapy response in different cancer systems, offering a versatile and cost-effective approach for assessing therapy efficacy in three-dimensional culture models.

Characterization of Polarimetric Properties in Various Brain Tumor Types Using Wide-Field Imaging Mueller Polarimetry.

Neuro-oncological surgery is the primary brain cancer treatment, yet it faces challenges with gliomas due to their invasiveness and the need to preserve neurological function. Hence, radical resection is often unfeasible, highlighting the importance of precise tumor margin delineation to prevent neurological deficits and improve prognosis. Imaging Mueller polarimetry, an effective modality in various organ tissues, seems a promising approach for tumor delineation in neurosurgery. To further assess its use, we characterized the polarimetric properties by analysing 45 polarimetric measurements of 27 fresh brain tumor samples, including different tumor types with a strong focus on gliomas. Our study integrates a wide-field imaging Mueller polarimetric system and a novel neuropathology protocol, correlating polarimetric and histological data for accurate tissue identification. An image processing pipeline facilitated the alignment and overlay of polarimetric images and histological masks. Variations in depolarization values were observed for grey and white matter of brain tumor tissue, while differences in linear retardance were seen only within white matter of brain tumor tissue. Notably, we identified pronounced optical axis azimuth randomization within tumor regions. This study lays the foundation for machine learning-based brain tumor segmentation algorithms using polarimetric data, facilitating intraoperative diagnosis and decision making.

Reporting of somatic variants in clinical cancer care: recommendations of the Swiss Society of Molecular Pathology.

Somatic variant testing through next-generation sequencing (NGS) is well integrated into Swiss molecular pathology laboratories and has become a standard diagnostic method for numerous indications in cancer patient care. Currently, there is a wide variation in reporting practices within our country, and as patients move between different hospitals, it is increasingly necessary to standardize NGS reports to ease their reinterpretation. Additionally, as many different stakeholders-oncologists, hematologists, geneticists, pathologists, and patients-have access to the NGS report, it needs to contain comprehensive and detailed information in order to answer the questions of experts and avoid misinterpretation by non-experts. In 2017, the Swiss Institute of Bioinformatics conducted a survey to assess the differences in NGS reporting practices across ten pathology institutes in Switzerland. The survey examined 68 reporting items and identified 48 discrepancies. Based on these findings, the Swiss Society of Molecular Pathology initiated a Delphi method to reach a consensus on a set of recommendations for NGS reporting. Reports should include clinical information about the patient and the diagnosis, technical details about the sample and the test performed, and a list of all clinically relevant variants and variants of uncertain significance. In the absence of a consensus on an actionability scheme, the five-class pathogenicity scheme proposed by the ACMG/AMP guideline must be included in the reports. The Swiss Society of Molecular Pathology recognizes the importance of including clinical actionability in the report and calls on the European community of molecular pathologists and oncologists to reach a consensus on this issue.

Rosette-forming glioneuronal tumor of the cerebellum in statu nascendi: an incidentally detected diminutive example indicates derivation from the internal granule cell layer.

Rosette-forming glioneuronal tumor (RGNT) is a recently introduced, indolent neoplasm composed of diminutive circular aggregates of neurocytic-like cells on a noninfiltrative astrocytic background, typically located in the cerebellar midline The traded concept of RGNT being derived from site-specific periventricular precursors may be questioned in the face of extracerebellar examples as well as ones occurring in combination with other representatives of the glioneuronal family. We describe a hitherto not documented example of asymptomatic RGNT discovered during autopsy of a 74-year-old male. Located in the tuberal vermis, this lesion of 6 mm diameter consisted of several microscopic nests of what were felt to represent nascent stages of RGNT, all of them centered on the internal granular layer, and ranging from mucoid dehiscences thereof to fully evolved - if small - tumor foci. Molecular genetic analysis revealed a missense mutation in Exon 20 of the PIK3CA gene involving an A→G transition at Nucleotide 3140. On the other hand, neither codeletion of chromosomes 1p/19q nor pathogenic mutations of IDH1/2 were detected. By analogy with in situ paradigms in other organs, we propose that this tumor is likely to have arisen from the internal granular layer, rather than the plate of the 4th ventricle. A suggestive departure from the wholesale argument of "undifferentiated precursors", this finding also indirectly indicates that a subset of non-classical RGNTs - in particular extracerebellar examples, whose origin cannot be mechanistically accounted for by either of the above structures - may possibly reflect an instance of phenotypic convergence, rather than a lineage-restricted entity.

Evaluation of MTAP and p16 immunohistochemical deficiency as surrogate marker for CDKN2A/B homozygous deletion in gliomas.

Homozygous deletion (HD) of the CDKN2A/B locus has emerged as an unfavourable prognostic marker in diffuse gliomas, both IDH-mutant and IDH-wild-type. Testing for CDKN2A/B deletions can be performed by a variety of approaches, including copy number variation (CNV) analysis based on gene array analysis, next generation sequencing (NGS) or fluorescence in situ hybridisation (FISH), but questions remain regarding the accuracy of testing modalities. In this study, we assessed: (1) the utility of S-methyl-5'-thioadenosine phosphorylase (MTAP) and cellular tumour suppressor protein pl61NK4a (p16) immunostainings as surrogate markers for CDKN2A/B HD in gliomas, and (2) the prognostic value of MTAP, across different histological tumour grades and IDH mutation status. One hundred consecutive cases of diffuse and circumscribed gliomas (Cohort 1) were collected, in order to correlate MTAP and p16 expression with the CDKN2A/B status in the CNV plot of each tumour. IDH1 R132H, ATRX and MTAP immunohistochemistry was performed on next generation tissue microarrays (ngTMAs) of 251 diffuse gliomas (Cohort 2) for implementing survival analysis. Complete loss of MTAP and p16 by immunohistochemistry was 100% and 90% sensitive as well as 97% and 89% specific for CDKN2A/B HD, respectively, as identified on CNV plot. Only two cases (2/100) with MTAP and p16 loss of expression did not demonstrate CDKN2A/B HD in CNV plot; however, FISH analysis confirmed the HD for CDKN2A/B. Moreover, MTAP deficiency was associated with shortened survival in IDH-mutant astrocytomas (n=75; median survival 61 vs 137 months; p<0.0001), IDH-mutant oligodendrogliomas (n=59; median survival 41 vs 147 months; p<0.0001) and IDH-wild-type gliomas (n=117; median survival 13 vs 16 months; p=0.011). In conclusion, MTAP immunostaining is an important complement for diagnostic work-up of gliomas, because of its excellent correlation with CDKN2A/B status, robustness, rapid turnaround time and low costs, and provides significant prognostic value in IDH-mutant astrocytomas and oligodendrogliomas, while p16 should be used cautiously.

Cribriform Morular Thyroid Carcinoma - Ultimobranchial Pouch-Related? Deep Molecular Insights of a Unique Case.

A 44-year-old female patient with a familial adenomatous polyposis (FAP) was diagnosed with a cribriform morular thyroid carcinoma (CMTC). We observed within the very necrotic tumor a small but distinct poorly differentiated carcinomatous component. As expected, next generation sequencing of both components revealed a homozygous APC mutation and in addition, a TERT promoter mutation. A TP53 mutation was found exclusively in the CMTC part, while the poorly differentiated component showed a clonal evolution, harboring an activating PIK3CA mutation and copy number gains of BRCA2, FGF23, FGFR1, and PIK3CB-alterations which are typically seen in squamous cell carcinoma. The mutational burden in both components was low, and there was no evidence for microsatellite instability. No mutations involving the mitogen-activated protein kinase (MAPK) pathway, typically seen in papillary thyroid carcinomas, were detected. Immunohistochemically, all tumor parts were negative for thyroglobulin, providing further evidence that this entity does not belong to the follicular epithelial cell-derived thyroid carcinoma group. CD5 was negative in the poorly differentiated component, making a relation to intrathyroidal thymic carcinoma rather unlikely. However, since this marker was seen in the morules, a loss in the poorly differentiated component and a relation to the ultimobranchial body cannot be excluded either. After total thyroidectomy and radioiodine ablation, the patient was disease-free with no residual tumor burden on 2-year follow-up.

Effects of formalin fixation on polarimetric properties of brain tissue: fresh or fixed?

Significance: Imaging Mueller polarimetry (IMP) appears as a promising technique for real-time delineation of healthy and neoplastic tissue during neurosurgery. The training of machine learning algorithms used for the image post-processing requires large data sets typically derived from the measurements of formalin-fixed brain sections. However, the success of the transfer of such algorithms from fixed to fresh brain tissue depends on the degree of alterations of polarimetric properties induced by formalin fixation (FF). Aim: Comprehensive studies were performed on the FF induced changes in fresh pig brain tissue polarimetric properties. Approach: Polarimetric properties of pig brain were assessed in 30 coronal thick sections before and after FF using a wide-field IMP system. The width of the uncertainty region between gray and white matter was also estimated. Results: The depolarization increased by 5% in gray matter and remained constant in white matter following FF, whereas the linear retardance decreased by 27% in gray matter and by 28% in white matter after FF. The visual contrast between gray and white matter and fiber tracking remained preserved after FF. Tissue shrinkage induced by FF did not have a significant effect on the uncertainty region width. Conclusions: Similar polarimetric properties were observed in both fresh and fixed brain tissues, indicating a high potential for transfer learning.

Integrated longitudinal analysis of adult grade 4 diffuse gliomas with long-term relapse interval revealed upregulation of TGF-ß signaling in recurrent tumors.

BACKGROUND: Adult-type diffuse gliomas, CNS WHO grade 4 are the most aggressive primary brain tumors and represent a particular challenge for therapeutic intervention. METHODS: In a single-center retrospective study of matched pairs of initial and post-therapeutic glioma cases with a recurrence period greater than 1 year, we performed whole exome sequencing combined with mRNA and microRNA expression profiling to identify processes that are altered in recurrent gliomas. RESULTS: Mutational analysis of recurrent gliomas revealed early branching evolution in 75% of the patients. High plasticity was confirmed at the mRNA and miRNA levels. SBS1 signature was reduced and SBS11 was elevated, demonstrating the effect of alkylating agent therapy on the mutational landscape. There was no evidence for secondary genomic alterations driving therapy resistance. ALK7/ACVR1C and LTBP1 were upregulated, whereas LEFTY2 was downregulated, pointing towards enhanced Tumor Growth Factor ß (TGF-ß) signaling in recurrent gliomas. Consistently, altered microRNA expression profiles pointed towards enhanced Nuclear Factor Kappa B and Wnt signaling that, cooperatively with TGF-ß, induces epithelial to mesenchymal transition (EMT), migration, and stemness. TGF-ß-induced expression of pro-apoptotic proteins and repression of antiapoptotic proteins were uncoupled in the recurrent tumor. CONCLUSIONS: Our results suggest an important role of TGF-ß signaling in recurrent gliomas. This may have clinical implications since TGF-ß inhibitors have entered clinical phase studies and may potentially be used in combination therapy to interfere with chemoradiation resistance. Recurrent gliomas show high incidence of early branching evolution. High tumor plasticity is confirmed at the level of microRNA and mRNA expression profiles.

Pleiotropy of PP2A Phosphatases in Cancer with a Focus on Glioblastoma IDH Wildtype.

Serine/Threonine protein phosphatase 2A (PP2A) is a heterotrimeric (or occasionally, heterodimeric) phosphatase with pleiotropic functions and ubiquitous expression. Despite the fact that they all contribute to protein dephosphorylation, multiple PP2A complexes exist which differ considerably by their subcellular localization and their substrate specificity, suggesting diverse PP2A functions. PP2A complex formation is tightly regulated by means of gene expression regulation by transcription factors, microRNAs, and post-translational modifications. Furthermore, a constant competition between PP2A regulatory subunits is taking place dynamically and depending on the spatiotemporal circumstance; many of the integral subunits can outcompete the rest, subjecting them to proteolysis. PP2A modulation is especially important in the context of brain tumors due to its ability to modulate distinct glioma-promoting signal transduction pathways, such as PI3K/Akt, Wnt, Ras, NF-κb, etc. Furthermore, PP2A is also implicated in DNA repair and survival pathways that are activated upon treatment of glioma cells with chemo-radiation. Depending on the cancer cell type, preclinical studies have shown some promise in utilising PP2A activator or PP2A inhibitors to overcome therapy resistance. This review has a special focus on "glioblastoma, IDH wild-type" (GBM) tumors, for which the therapy options have limited efficacy, and tumor relapse is inevitable.

Programmed Death-Ligand 1 Expression in Lung Cancer and Paired Brain Metastases-a Single-Center Study in 190 Patients.

Introduction: Expression of programmed death-ligand 1 (PD-L1) is the only routinely used tissue biomarker for predicting response to programmed cell death protein 1/PD-L1 inhibitors. It is to date unclear whether PD-L1 expression is preserved in brain metastases (BMs). Methods: In this single-center, retrospective study, we evaluated PD-L1 expression using the SP263 assay in consecutively resected BMs of lung carcinomas and paired primary tumors, diagnosed from 2000 to 2015, with correlation to clinicopathological and molecular tumor and patient characteristics. Results: PD-L1 tumor proportional score (TPS) could be evaluated on whole tissue slides in 191 BMs and 84 paired primary lung carcinomas. PD-L1 TPS was less than 1% in 113 of 191 (59.2%), 1% to 49% in 34 of 191 (17.8%), and greater than or equal to 50% in 44 of 191 (23.0%) BMs. TPS was concordant between BMs and paired primary lung carcinomas in most cases, with discordance regarding the clinically relevant cutoffs at 1% and 50% in 18 of 84 patients (21.4%). Four of 18 discordant cases had no shared mutations between the primary lung carcinoma and BM. Intratumoral heterogeneity, as assessed using tissue microarray cores, was only significant at the primary site (p Wilcoxon signed rank = 0.002) with higher PD-L1 TPS at the infiltration front (mean = 40.4%, interquartile range: 0%-90%). Neither TPS greater than or equal to 1% nor TPS greater than or equal to 50% nor discordance between the primary lung carcinoma and BMs had prognostic significance regarding overall survival or BM-specific overall survival. Conclusions: PD-L1 expression was mostly concordant between primary lung carcinoma and its BM and between resections of BM and stereotactic biopsies, mirrored by tissue microarray cores. Differences in PD-L1 TPS existed primarily in cases with TPS greater than 10%, for which also human assessment tends to be most error prone.