BACKGROUND: Diesel exhaust particles (DEP), which contain hazardous compounds, are emitted during the combustion of diesel. As approximately one-third of the vehicles worldwide use diesel, there are growing concerns about the risks posed by DEP to human health. Long-term exposure to DEP is associated with airway hyperresponsiveness, pulmonary fibrosis, and inflammation; however, the molecular mechanisms behind the effects of DEP on the respiratory tract are poorly understood. Such mechanisms can be addressed by examining transcriptional and DNA methylation changes. Although several studies have focused on the effects of short-term DEP exposure on gene expression, research on the transcriptional effects and genome-wide DNA methylation changes caused by long-term DEP exposure is lacking. Hence, in this study, we investigated transcriptional and DNA methylation changes in human adenocarcinoma alveolar basal epithelial A549 cells caused by prolonged exposure to DEP and determined whether these changes are concordant.
RESULTS: DNA methylation analysis using the Illumina Infinium MethylationEPIC BeadChips showed that the methylation levels of DEP-affected CpG sites in A549 cells changed in a dose-dependent manner; the extent of change increased with increasing dose reaching the statistical significance only in samples exposed to 30 µg/ml DEP. Four-week exposure to 30 µg/ml of DEP significantly induced DNA hypomethylation at 24,464 CpG sites, which were significantly enriched for DNase hypersensitive sites, genomic regions marked by H3K4me1 and H3K27ac, and several transcription factor binding sites. In contrast, 9,436 CpG sites with increased DNA methylation levels were significantly overrepresented in genomic regions marked by H3K27me3 as well as H3K4me1 and H3K27ac. In parallel, gene expression profiling by RNA sequencing demonstrated that long-term exposure to DEP altered the expression levels of 2,410 genes, enriching 16 gene sets including Xenobiotic metabolism, Inflammatory response, and Senescence. In silico analysis revealed that the expression levels of 854 genes correlated with the methylation levels of the DEP-affected cis-CpG sites.
CONCLUSIONS: To our knowledge, this is the first report of genome-wide transcriptional and DNA methylation changes and their associations in A549 cells following long-term exposure to DEP.

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In recent years, the classification of adult-type diffuse gliomas has undergone a revolution, wherein specific molecular features now represent defining diagnostic criteria of IDH-wild-type glioblastomas, IDH-mutant astrocytomas, and IDH-mutant 1p/19q-codeleted oligodendrogliomas. With the introduction of the 2021 WHO CNS classification, additional molecular alterations are now integrated into the grading of these tumors, given equal weight to traditional histologic features. However, there remains a great deal of heterogeneity in patient outcome even within these established tumor subclassifications that is unexplained by currently codified molecular alterations, particularly in the IDH-mutant astrocytoma category. There is also significant intercellular genetic and epigenetic heterogeneity and plasticity with resulting phenotypic heterogeneity, making these tumors remarkably adaptable and robust, and presenting a significant barrier to the design of effective therapeutics. Herein, we review the mechanisms and consequences of genetic and epigenetic instability, including chromosomal instability (CIN), microsatellite instability (MSI)/mismatch repair (MMR) deficits, and epigenetic instability, in the underlying biology, tumorigenesis, and progression of IDH-mutant astrocytomas. We also discuss the contribution of recent high-resolution transcriptomics studies toward defining tumor heterogeneity with single-cell resolution. While intratumoral heterogeneity is a well-known feature of diffuse gliomas, the contribution of these various processes has only recently been considered as a potential driver of tumor aggressiveness. CIN has an independent, adverse effect on patient survival, similar to the effect of histologic grade and homozygous CDKN2A deletion, while MMR mutation is only associated with poor overall survival in univariate analysis but is highly correlated with higher histologic/molecular grade and other aggressive features. These forms of genomic instability, which may significantly affect the natural progression of these tumors, response to therapy, and ultimately clinical outcome for patients, are potentially measurable features which could aid in diagnosis, grading, prognosis, and development of personalized therapeutics.

EGFR aberrations are reported in a subset of myofibroblastic lesions with kinase domain duplication (EGFR-KDD) and exon 20 mutations being assigned to infantile fibrosarcomas (IFS), mesoblastic nephroma, and fibrous hamartoma of infancy (FHI), respectively. In this retrospective study, we correlated molecular findings with the histomorphology of 14 myofibroblastic lesions harboring such genetic changes identified by NGS. We additionally performed DNA methylation profiling (DNAmp) and immunohistochemistry. Lesions were from 10 males and 4 females with a mean age of 3 years (range, 0.3-14) and occurred subcutaneously in the upper limbs (n = 5), lower limbs (n = 3), back/thorax (n = 5), and the nasal cavity (n = 1). Eleven were cured by surgery, including 1 relapsed case. Two patients were lost to follow-up. One case was very recent, and the patient was biopsied. Histologically, the lesions showed a wide spectrum varying from classic FHI (n = 9) to IFS (n = 1) or lipofibromatosis-like tumors (LFT-like) (n = 2) or dermatofibrosarcoma protuberans-like (DFSP-like) (n = 1) to a predominantly myxoid spindle cell lesion (n = 1). Immunohistochemically, all neoplasms stained with CD34, whereas S100 was positive in 2/14. EGFR expression was observed in 9/10 cases. Molecularly, the IFS and 1 LFT-like harbored EGFR-KDD, whereas an exon 20 mutation was identified in all FHI, 1 LFT-like, the DFSP-like, and in predominant myxoid spindle cell lesion. By DNAmp, all but 2 cases formed a well-defined cluster, demonstrating that these lesions are also epigenetically related. In conclusion, EGFR kinase domain aberrations found in FHI, IFS, LFT-like, DFSP-like, and a spindle cell lesion with a predominant myxoid stroma of children and adolescents showed that these neoplasms with a broad morphologic spectrum belong to the group of protein kinase-related lesions with a distinct epigenetic signature. Molecular analyses, including DNAmp, help to identify and characterize this emerging category and become mandatory when targeted treatment is considered.

It is now understood that identical gene fusions may be shared by different entities. We report a distinctive neoplasm of the skin and subcutis, harboring the Ewing sarcoma-associated EWSR1::FLI1 fusion but differing otherwise from Ewing sarcoma. Slides and blocks for 5 cutaneous neoplasms coded as other than Ewing sarcoma and harboring EWSR1::FLI1 were retrieved. Immunohistochemical and molecular genetic results were abstracted from reports. Methylation profiling was performed. Clinical information was obtained. The tumors occurred in 4 men and 1 woman (median: 25 years of age; range: 19-69 years) and involved the skin/subcutis of the back (2), thigh, buttock, and chest wall (median: 2.4 cm; range: 1-11 cm). Two tumors were present \"years\" before coming to clinical attention. The lesions were multinodular and circumscribed and consisted of nests of bland, round cells admixed with hyalinized collagenous bands containing spindled cells. Hemorrhage and cystic change were often present; necrosis was absent. All were diffusely S100 protein/SOX10-positive; 4 of 5 were CD99-negative. One tested case was strongly positive for NKX2.2. A variety of other tested markers were either focally positive (glial fibrillary acidic protein, p63) or negative. Molecular genetic results were as follows: EWSR1 exon 7::FLI1 exon 8, EWSR1 exon 11::FLI1 exon 5, EWSR1 exon 11::FLI1 exon 6, EWSR1 exon 7::FLI1 exon 6, and EWSR1 exon 10::FLI1 exon 6. Methylation profiling (3 cases) showed these to form a unique cluster, distinct from Ewing sarcoma. All patients underwent excision with negative margins; one received 1 cycle of chemotherapy. Clinical follow-up showed all patients to be alive without disease (median: 17 months; range: 11-62 months). Despite similar gene fusions, the morphologic, immunohistochemical, epigenetic, and clinical features of these unique EWSR1::FLI1-fused neoplasms of the skin and subcutis differ substantially from Ewing sarcoma. Interestingly, EWSR1 rearrangements involved exons 10 or 11, only rarely seen in Ewing sarcoma, in a majority of cases. Superficial neurocristic EWSR1::FLI1 fusion tumors should be rigorously distinguished from true cutaneous Ewing sarcomas.

BACKGROUND: Primary spinal cord tumors, especially primary spinal cord glioblastoma multiforme (PSC-GBM), are exceptionally rare, accounting for less than 1.5% of all spinal tumors. Their infrequency and aggressive yet atypical presentation make diagnosis challenging. In uncertain cases, a surgical approach for tissue diagnosis is often optimal.
METHODS: A 76-year-old male presented with a rapidly progressing clinical history marked by worsening extremity weakness, urinary retention, and periodic fecal incontinence alongside diffuse changes on neuraxis imaging. The patient, in whom subacute polyneuropathy was initially diagnosed, received multiple rounds of steroids and intravenous immunoglobulin without clinical improvement. Histopathological review of the biopsy tissue yielded an initial diagnosis of spindle cell neoplasm. Next-generation sequencing (NGS) is done routinely on all neuropathology specimens at the authors\' institution, and methylation profiling is pursued in difficult cases. Ultimately, NGS and methylation profiling results were essential to an integrated final diagnosis of GBM.
CONCLUSIONS: PSC-GBM is a rare but highly aggressive occurrence of this tumor. Prolonged back pain, rapid neurological decline, and imaging changes warrant the consideration of lesional biopsy for precise disease characterization. In inconclusive cases, NGS has proved invaluable for clinical clarification and diagnosis, underscoring its importance for integrated diagnoses in guiding appropriate treatment strategies.

Alterations in kinase genes such as NTRK1/2/3, RET, and BRAF underlie infantile fibrosarcoma (IFS), the emerging entity \'NTRK-rearranged spindle cell neoplasms\' included in the latest WHO classification, and a growing set of tumors with overlapping clinical and pathological features. In this study, we conducted a comprehensive clinicopathological and molecular analysis of 22 cases of IFS and other kinase gene-altered spindle cell neoplasms affecting both pediatric and adult patients. Follow-up periods for 16 patients ranged in length from 10 to 130 months (mean 38 months). Six patients were treated with targeted therapy, achieving a partial or complete response in five cases. Overall, three cases recurred and one metastasized. Eight patients were free of disease, five were alive with disease, and two patients died. All cases showed previously reported morphological patterns. Based on the cellularity and level of atypia, cases were divided into three morphological grade groups. S100 protein and CD34 were at least focally positive in 12/22 and 14/22 cases, respectively. Novel PWWP2A::RET, NUMA1::RET, ITSN1::RAF1, and CAPZA2::MET fusions, which we report herein in mesenchymal tumors for the first time, were detected by RNA sequencing. Additionally, the first uterine case with BRAF and EGFR mutations and CD34 and S100 co-expression is described. DNA sequencing performed in 13 cases uncovered very rare additional genetic aberrations. The CNV profiles showed that high-grade tumors demonstrate a significantly higher percentage of copy number gains and losses across the genome compared with low- and intermediate-grade tumors. Unsupervised clustering of the tumors\' methylation profiles revealed that in 8/9 cases, the methylation profiles clustered with the IFS methylation class, irrespective of their clinicopathological or molecular features. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Small cell osteosarcoma (SCOS), a variant of conventional high-grade osteosarcoma (COS), may mimic fusion-driven round cell sarcomas (FDRCS) by overlapping clinico-radiological and histomorphological/immunohistochemical characteristics, hampering accurate diagnosis and consequently proper therapy. We retrospectively analyzed decalcified formalin-fixed paraffin-embedded (FFPE) samples of 18 bone tumors primarily diagnosed as SCOS by methylation profiling, fusion gene analysis, and immunohistochemistry.In eight cases, the diagnosis of SCOS was maintained, and in 10 cases it was changed into FDRCS, including three Ewing sarcomas (EWSR1::FLI1 in two cases and no identified fusion gene in the third case), two sarcomas with BCOR alterations (KMT2D::BCOR, CCNB3::BCOR, respectively), three mesenchymal chondrosarcomas (HEY1::NCOA2 in two cases and one case with insufficient RNA quality), and two sclerosing epithelioid fibrosarcomas (FUS::CREBL3 and EWSR1 rearrangement, respectively).Histologically, SCOS usually possessed more pleomorphic cells in contrast to the FDRCS showing mainly monomorphic cellular features. However, osteoid was seen in the latter tumors as well, often associated with slight pleomorphism. Also, the immunohistochemical profile (CD99, SATB2, and BCOR) overlapped.Clinically and radiologically, similarities between SCOS and FDRCS were observed, with by imaging only minimal presence or lack of (mineralized) osteoid in most of the SCOSs.In conclusion, discrimination of SCOS, epigenetically related to COS, versus FDRCS of bone can be challenging but is important due to different biology and therefore therapeutic strategies. Methylation profiling is a reliable and robust diagnostic test especially on decalcified FFPE material. Subsequent fusion gene analysis and/or use of specific immunohistochemical surrogate markers can be used to substantiate the diagnosis.

Since the introduction of new molecular techniques, the diagnostic landscape of soft tissue and bone tumors has expanded greatly over the past few years. The use of new molecular techniques has led to the identification of new genetic alterations and, therefore, to a better understanding of tumorigenesis, tumor detection and classification. Furthermore, methylation profiling has emerged as a classification tool for soft tissue and bone tumors. Molecular pathology also plays an important role in the determination of patient prognosis and in the identification of targets that can be used for targeted therapy. As a result, molecular pathology has gained a more prominent role in the daily practice of the surgical pathologist. This review delves into various molecular techniques applied in the surgical pathology of soft tissue and bone tumors. It highlights their applications through the analysis of five specific cases.

This paper examines the link between CNS tumor biology and heterogeneity and the use of genome-wide DNA methylation profiling as a clinical diagnostic platform. CNS tumors are the most common solid tumors in children, and their prognosis remains poor. This study retrospectively analyzed pediatric patients with CNS embryonal tumors in Hong Kong between 1999 and 2017, using data from the territory-wide registry and available formalin-fixed paraffin-embedded tumor tissue. After processing archival tumor tissue via DNA extraction, quantification, and methylation profiling, the data were analyzed by using the web-based DKFZ classifier (Molecular Neuropathology (MNP) 2.0 v11b4) and t-SNE analysis. Methylation profiles were deemed informative in 85 samples. Epigenetic data allowed molecular subgrouping and confirmed diagnosis in 65 samples, verified histologic diagnosis in 8, and suggested an alternative diagnosis in 12. This study demonstrates the potential of DNA methylation profiling in characterizing pediatric CNS embryonal tumors in a large cohort from Hong Kong, which should enable regional and international collaboration in future pediatric neuro-oncology research.