Targeted inhibition of the methyltransferase SETD8 synergizes with the Wee1 inhibitor adavosertib in restraining glioblastoma growth.

Despite intense research efforts, glioblastoma remains an incurable brain tumor with a dismal median survival time of 15 months. Thus, identifying new therapeutic targets is an urgent need. Here, we show that the lysine methyltransferase SETD8 is overexpressed in 50% of high-grade gliomas. The small molecule SETD8 inhibitor UNC0379, as well as siRNA-mediated inhibition of SETD8, blocked glioblastoma cell proliferation, by inducing DNA damage and activating cell cycle checkpoints. Specifically, in p53-proficient glioblastoma cells, SETD8 inhibition and DNA damage induced p21 accumulation and G1/S arrest whereas, in p53-deficient glioblastoma cells, DNA damage induced by SETD8 inhibition resulted in G2/M arrest mediated by Chk1 activation. Checkpoint abrogation, by the Wee1 kinase inhibitor adavosertib, induced glioblastoma cell lines and primary cells, DNA-damaged by UNC0379, to progress to mitosis where they died by mitotic catastrophe. Finally, UNC0379 and adavosertib synergized in restraining glioblastoma growth in a murine xenograft model, providing a strong rationale to further explore this novel pharmacological approach for adjuvant glioblastoma treatment.

Clinical, histopathological and molecular features of dedifferentiated melanomas: An EORTC Melanoma Group Retrospective Analysis.

PURPOSE: Dedifferentiated melanoma (DedM) poses significant diagnostic challenges. We aimed to investigate the clinical, histopathological and molecular features of DedM. Methylation signature (MS) and copy number profiling (CNP) were carried out in a subgroup of cases. PATIENTS AND METHODS: A retrospective series of 78 DedM tissue samples from 61 patients retrieved from EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group centres were centrally reviewed. Clinical and histopathological features were retrieved. In a subgroup of patients, genotyping through Infinium Methylation microarray and CNP analysis was carried out. RESULTS: Most patients (60/61) had a metastatic DedM showing most frequently an unclassified pleomorphic, spindle cell, or small round cell morphology akin to undifferentiated soft tissue sarcoma, rarely associated with heterologous elements. Overall, among 20 successfully analysed tissue samples from 16 patients, we found retained melanoma-like MS in only 7 tissue samples while a non-melanoma-like MS was observed in 13 tissue samples. In two patients from whom multiple specimens were analysed, some of the samples had a preserved cutaneous melanoma MS while other specimens exhibited an epigenetic shift towards a mesenchymal/sarcoma-like profile, matching the histological features. In these two patients, CNP was largely identical across all analysed specimens, in line with their common clonal origin, despite significant modification of their epigenome. CONCLUSIONS: Our study further highlights that DedM represents a real diagnostic challenge. While MS and genomic CNP may help pathologists to diagnose DedM, we provide proof-of-concept that dedifferentiation in melanoma is frequently associated with epigenetic modifications.

Genetic and epigenetic analysis of hepatocellular adenomas with atypical morphological features.

BACKGROUND: Hepatocellular adenoma (HCA) is a rare liver tumour, which can have atypical morphological features such as cytological atypia, pseudoglandular architecture, and altered reticulin framework. Little is known about the genetic and epigenetic alterations of such HCAs and whether they show the alterations classically found in hepatocellular carcinoma (HCC) or in HCA without atypical morphology. METHODS: We analysed five HCAs with atypical morphological features and one HCA with transition to HCC. Every tumour was subtyped by immunohistochemistry, sequenced by a targeted NGS panel, and analysed on a DNA methylation microarray. RESULTS: Subtyping of the five HCAs with atypical features revealed two ß-catenin mutated HCA (b-HCA), two ß-catenin mutated inflammatory HCA (b-IHCA), and one sonic hedgehog activated HCA (shHCA). None of them showed mutations typically found in HCC, such as, e.g. TERT or TP53 mutations. The epigenomic pattern of HCAs with atypical morphological features clustered with reference data for HCAs without atypical morphological features but not with HCC. Similarly, phyloepigenetic trees using the DNA methylation data reproducibly showed that HCAs with morphological atypia are much more similar to nonmalignant samples than to malignant samples. Finally, atypical HCAs showed no relevant copy number variations (CNV). CONCLUSION: In our series, mutational, DNA methylation, as well as CNV analyses, supported a relationship of atypical HCAs with nonatypical HCAs rather than with HCC. Therefore, in cases with difficult differential diagnosis between HCC and HCA, it might be advisable to perform targeted sequencing and/or combined methylation/copy number profiling.

An Integrated Epigenomic and Genomic View on Phyllodes and Phyllodes-like Breast Tumors.

Fibroepithelial lesions (FL) of the breast, in particular, phyllodes tumors (PT) and fibroadenomas, pose a significant diagnostic challenge. There are no generally accepted criteria that distinguish benign, borderline, malignant PT and fibroadenomas. Combined genome-wide DNA methylation and copy number variant (CNV) profiling is an emerging strategy to classify tumors. We compiled a series of patient-derived archival biopsy specimens reflecting the FL spectrum and histological mimickers including clinical follow-up data. DNA methylation and CNVs were determined by well-established microarrays. Comparison of the patterns with a pan-cancer dataset assembled from public resources including "The Cancer Genome Atlas" (TCGA) and "Gene Expression Omnibus" (GEO) suggests that FLs form a methylation class distinct from both control breast tissue as well as common breast cancers. Complex CNVs were enriched in clinically aggressive FLs. Subsequent fluorescence in situ hybridization (FISH) analysis detected respective aberrations in the neoplastic mesenchymal component of FLs only, confirming that the epithelial component is non-neoplastic. Of note, our approach could lead to the elimination of the diagnostically problematic category of borderline PT and allow for optimized prognostic patient stratification. Furthermore, the identified recurrent genomic aberrations such as 1q gains (including MDM4), CDKN2a/b deletions, and EGFR amplifications may inform therapeutic decision-making.

Evaluation of MGMT Gene Methylation in Neuroendocrine Neoplasms.

Unresectable neuroendocrine neoplasms (NENs) often poorly respond to standard therapeutic approaches. Alkylating agents, in particular temozolomide, commonly used to treat high-grade brain tumors including glioblastomas, have recently been tested in advanced or metastatic NENs, where they showed promising response rates. In glioblastomas, prediction of response to temozolomide is based on the assessment of the methylation status of the MGMT gene, as its product, O 6-methylguanine-DNA methyltransferase, may counteract the damaging effects of the alkylating agent. However, in NENs, such a biomarker has not been validated yet. Thus, we have investigated MGMT methylation in 42 NENs of different grades and from various sites of origin by two different approaches: in contrast to methylation-specific PCR (MSP), which is commonly used in glioblastoma management, amplicon bisulfite sequencing (ABS) is based on high-resolution, next-generation sequencing and interrogates several additional CpG sites compared to those covered by MSP. Overall, we found MGMT methylation in 74% (31/42) of the NENs investigated. A higher methylation degree was observed in well-differentiated tumors and in tumors originating in the gastrointestinal tract. Comparing MSP and ABS results, we demonstrate that the region analyzed by the MSP test is sufficiently informative of the MGMT methylation status in NENs, suggesting that this predictive parameter could routinely be interrogated also in NENs.

Integrated Analysis Of Immunotherapy Treated Clear Cell Renal Cell Carcinomas: An Exploratory Study.

Molecular or immunological differences between responders and nonresponders to immune checkpoint inhibitors (ICIs) of clear cell renal cell carcinomas (ccRCCs) remain incompletely understood. To address this question, we performed next-generation sequencing, methylation analysis, genome wide copy number analysis, targeted RNA sequencing and T-cell receptor sequencing, and we studied frequencies of tumor-infiltrating CD8+ T cells, presence of tertiary lymphoid structures (TLS) and PD-L1 expression in 8 treatment-naive ccRCC patients subsequently treated with ICI (3 responders, 5 nonresponders). Unexpectedly, we identified decreased frequencies of CD8+ tumor-infiltrating T cells and TLS, and a decreased expression of PD-L1 in ICI responders when compared with nonresponders. However, neither tumor-specific genetic alterations nor gene expression profiles correlated with response to ICI or the observed immune features. Our results underline the challenge to stratify ccRCC patients for immunotherapy based on routinely available pathologic primary tumor material, even with advanced technologies. Our findings emphasize the analysis of pretreated metastatic tissue in line with recent observations describing treatment effects on the tumor microenvironment. In addition, our data call for further investigation of additional parameters in a larger ccRCC cohort to understand the mechanistic implications of the observed differences in tumor-infiltrating CD8+ T cells, TLS, and PD-L1 expression.

Differentiation of rare brain tumors through unsupervised machine learning: Clinical significance of in-depth methylation and copy number profiling illustrated through an unusual case of IDH wildtype glioblastoma.

Methylation profiling has become a mainstay in brain tumor diagnostics since the introduction of the first publicly available classification tool by the German Cancer Research Center in 2017. We demonstrate the capability of this system through an example of a rare case of IDH wildtype glioblastoma diagnosed in a patient previously treated for T-cell acute lymphoblastic leukemia. Our novel in-house diagnostic tool EpiDiP provided hints arguing against a radiation-induced tumor, identified a novel recurrent genetic aberration, and thus informed about a potential therapeutic target.

NUT midline carcinomas and their differentials by a single molecular profiling method: a new promising diagnostic strategy illustrated by a case report.

NUT midline carcinoma is an aggressive neoplasm defined by chromosomal rearrangements of the nuclear protein in testis (NUT) gene (NUTM1). In this article, we present a strategy to detect this rare tumor through a standard DNA methylation array analysis even when occurring in unusual anatomic sites. We illustrate our approach through a case study in which we detected metastatic spread of a NUT midline carcinoma within a bone marrow biopsy that exhibited histological features of a blastoid, undifferentiated neoplasm. Our strategy builds on molecular data derived from The Cancer Genome Atlas and Gene Expression Omnibus as well as computational strategies adopted from the Brain Tumor Methylation Classifier. It is a combined approach that detects the unusual cell lineage of NUT midline carcinomas and makes diagnostic use of the entity-specific copy number alterations.

Diagnosis of adult-onset MELAS syndrome in a 63-year-old patient with suspected recurrent strokes - a case report.

BACKGROUND: Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is a mitochondrial cytopathy caused by mutations in mitochondrial DNA. Clinical manifestation is typically before the age of 40. CASE PRESENTATION: We present the case of a 63-year-old female in whom the symptoms of MELAS were initially misdiagnosed as episodes of recurrent ischemic strokes. Brain imaging including MRI, clinical and laboratory findings that lent cues to the diagnosis of MELAS are discussed. In addition, MRI findings in MELAS in comparison to imaging mimics of MELAS are presented. CONCLUSIONS: This case underscores the importance of considering MELAS as a potential cause of recurrent stroke-like events if imaging findings are untypical for cerebral infarction, even among middle-aged patients with vascular risk factors.

Hymenolepis nana: A case report of a perfect IBD camouflage warrior.

RATIONALE: There is evidence that parasitic helminths can ameliorate colitis in animal models and humans. Although infections with Hymenolepis sp. are clinically benign, the immunomodulatory interactions between host and parasite are largely unknown. PATIENT CONCERNS: In this study we examined the intestinal mucosa of an adult asymptomatic patient harboring adult and larval dwarf tapeworms (Hymenolepis nana) who underwent surgery for an unrelated reason. INTERVENTIONS: Routine histology and immunohistochemistry were performed to characterize the host's response to the parasite. Parasitic DNA was sequenced to identify the tapeworm species. DIAGNOSES: Morphological and immunohistochemical studies showed a nearly complete absence of an anti-parasite host immune response. The outer surface of the parasite also showed prominent cross-reactivity with various tested leukocyte antigens. Our findings closely resemble experimentally obtained data from the H. diminuta-infected rat at the state of persistent colonization. OUTCOMES: Cross-reactivity of parasite-borne molecules with anti-human-leukocyte antibodies indicates a potential functional role in active modulation of the host's immune response. LESSIONS: We believe that better understanding of the host-cestode interaction will certainly extend our knowledge on auto-aggressive disorders such as inflammatory bowel disease and might provide potential treatment options.

Mitochondrial cytopathy with common MELAS mutation presenting as multiple system atrophy mimic.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome1 is one of the most frequently inherited mitochondrial disorders. MELAS syndrome is a systemic disease with multiple organ involvement.2 The most common mutation in MELAS is the m.3243A>G mutation in the MT-TL1 gene.2.

Rhabdoid large cell carcinoma of lung, with illustrative immunohistochemical and molecular findings.

Large cell carcinomas with rhabdoid phenotype (LCC-RP) account for <1% of pulmonary large cell carcinomas and are associated with extremely poor prognosis. We report a case of a 64-year-old male patient who presented at an advanced stage with a LCC-RP, arising from a poorly differentiated adenocarcinoma of the lung. Ninety percent of the tumor consisted of large pleomorphic rhabdoid tumor cells that showed eosinophilic cytoplasmic inclusions and the remaining 10% showed evidence of adenomatous differentiation. Rhabdoid areas were immunohistochemically positive for pan-cytokeratin AE1/3, epithelial membrane antigen, vimentin, thyroid transcription factor-1, integrase interactor-1, and negative for desmin. Nuclear positivity was absent for Myo-D1. The parent adenocarcinoma was positive for thyroid transcription factor-1 and cytokeratins 7. Epidermal growth factor receptor mutation analysis revealed the same mutation (p.delL747-T751) in both areas, suggesting that the malignant rhabdoid phenotype represents a dedifferentiation phenomenon of the adenocarcinoma. This is the first reported case of an Exon 19 deletion in epidermal growth factor receptor of the activating type detected in a LCC-RP associated with a poorly differentiated pulmonary adenocarcinoma.