Faculty Bibliography

Gynecologic neuroectodermal tumors either exhibit central nervous system (CNS) differentiation (CNS-like) or represent Ewing sarcoma (EWS) which lacks CNS features and harbors FET-ETS gene fusions. DNA methylation profiling reclassified CNS primitive neuroectodermal tumors into common CNS neoplasms or embryonal tumors with specific epigenetic/ genetic characteristics. Its utility in classifying gynecologic neuroectodermal tumors is unknown. Whole genome DNA methylation profiling was performed on 26 gynecologic neuroectodermal tumors (22 CNS-like tumors, 4 EWS) arising in the ovary, paratubal soft tissue, uterus, and vulva, which were classified by using sarcoma and CNS tumor DNA methylation classifiers. Sarcoma-related gene fusions were confirmed by fluorescence in situ hybridization (FISH) or targeted RNA next generation sequencing (NGS). Tumor only whole exome sequencing (WES) was performed in 13 cases. Copy number alterations and zygosity were inferred from DNA methylation array and WES data. Methylation abnormalities associated with imprinting were examined. The sarcoma methylation classifier identified EWS (n=3) and high-grade endometrial stromal sarcoma (n=1), confirmed by FISH or NGS detection of EWSR1 and YWHAE rearrangements, respectively. The remaining CNS-like tumors were classified by DNA methylation with positive/valid (n=4), indeterminate (n=9), and negative (n=9) scores at family level. Methylation subclasses included teratoma; embryonal tumor with multilayered rosettes, atypical; medulloblastoma, SHH-activated, subtype 3; medulloblastoma, group 3; intraocular medulloepithelioma; supratentorial ependymoma, ZFTA::RELA fused, subclass A; and diffuse pediatric-type high-grade glioma, MYCN subtype. Male gender was predicted in 54% of methylation-confirmed CNS-like tumors and none of the sarcomas. Among CNS-like tumors, copy number analyses identified genome-wide chromosomal gains and losses, and WES revealed genome-wide allelic imbalance suggestive of genome wide duplications. Epigenetic imprinting analyses showed increased paternal or maternal imprinting signal across multiple chromosomes suggesting uniparental duplication. DNA methylation profiling successfully classified gynecologic neuroectodermal tumors as known CNS tumor or sarcoma entities. Epigenetic and exomic studies suggest a male genome and increased maternal allelic contribution in CNS-like tumors, suggesting development via conception or chimerism.

PURPOSE/OBJECTIVE:Histopathological grading of IDH-mutant astrocytomas demonstrates limited prognostic accuracy. However, DNA methylation subclassification has demonstrated improved prognostication beyond histological grading. This study aimed to investigate the associations between imaging features, tumor volumetric data, and DNA methylation grade in IDH-mutant astrocytomas. METHODS:We analyzed imaging features and volumetric data for 72 patients diagnosed with IDH-mutant astrocytomas, who underwent preoperative MRI and DNA methylation profiling. VASARI features and multicompartmental volumetrics were evaluated. Logistic regression was used to identify imaging predictors of methylation subclass, WHO histologic grade, copy number variation (CNV), and CDKN2A/B homozygous deletion. Univariable and multivariable Cox proportional hazard models were also developed to assess these variables' influence on overall survival and progression-free survival. RESULTS:Patients were classified into 27 methylation high-grade (A_IDH_HG) and 45 methylation low-grade (A_IDH_LG) tumors. Tumor volumes and proportions varied by methylation grade, CNV status, and WHO histologic grade, but not by CDKN2A/B status. Imaging features distinguished methylation subclasses with 75% accuracy (AUC = 0.77). Methylation high-grade subclass was associated with imaging features such as midline crossing, ependymal extension, and poorly defined enhancing margins. Predictive performance for WHO histologic grade, CNV status, and CDKN2A/B deletion was moderate (AUC = 0.67, 0.69, and 0.65, respectively). Methylation grade, CDKN2A/B status, VASARI features, and proportions of edema and non-contrast enhancing tumor were significantly associated with survival. CONCLUSION/CONCLUSIONS:MRI-derived imaging features facilitate noninvasive prediction of DNA methylation subclass in IDH-mutant astrocytomas.

Renal neoplasms are morphologically and molecularly heterogeneous, with their diagnosis often hindered by interobserver variability and overlapping microscopic features. A subset of cases is unclassifiable despite immunohistochemical, mutation, and cytogenetic-based diagnostic workup. Through examination of the genome-wide DNA methylation signatures of over 2000 renal neoplasms, we identified 23 coherent groups that correlate with known neoplasm types and identified novel clinically relevant subtypes of existing neoplasm types. We used machine learning models to develop and validate a classifier trained on DNA methylation profiles of 1284 samples. The classifier was tested on an external data set of 287 renal neoplasms with >90% concordance between expected neoplasm type and high-score DNA methylation-based classification. Discordance between the original histologic label and methylation class led to potential reclassification of some cases. This work demonstrates proof of principle for the feasibility of a DNA methylation classifier as a clinically useful tool to assist in the diagnosis of renal neoplasms.

BACKGROUND:Pituitary neuroendocrine tumors (PitNETs) are the most common intracranial neuroendocrine tumors. PitNETs can be challenging to classify, and current recommendations include a large immunohistochemical panel to differentiate among 14 WHO-recognized categories. METHODS:In this study, we analyzed clinical, immunohistochemical and DNA methylation data of 118 PitNETs to develop a clinico-molecular approach to classifying PitNETs and identify epigenetic classes. RESULTS:CNS DNA methylation classifier has an excellent performance in recognizing PitNETs and distinguishing the three lineages when the calibrated score is ≥0.3. Unsupervised DNA methylation analysis separated PitNETs into two major clusters. The first was composed of silent gonadotrophs, which form a biologically distinct group of PitNETs characterized by clinical silencing, weak hormonal expression on immunohistochemistry, and simple copy number profile. The second major cluster was composed of corticotrophs and Pit1 lineage PitNETs, which could be further classified using DNA methylation into distinct subclusters that corresponded to clinically functioning and silent tumors and are consistent with transcription factor expression. Analysis of promoter methylation patterns correlated with lineage for corticotrophs and Pit1 lineage subtypes. However, the gonadotrophic genes did not show a distinct promoter methylation pattern in gonadotroph tumors compared to other lineages. Promoter of the NR5A1 gene, which encodes SF1, was hypermethylated across all PitNETs clinical and molecular subtypes including gonadotrophs with strong SF1 protein expression indicating alternative epigenetic regulation. CONCLUSION/CONCLUSIONS:Our findings suggest that classification of PitNETs may benefit from DNA methylation for clinicopathological stratification.

BACKGROUND:Meningiomas are the most common primary intracranial neoplasms, with highly variable patient outcomes. While most meningiomas are benign, a significant subset recurs postoperatively, presenting substantial treatment challenges. BAP1 gene inactivation has been suggested as a marker for aggressive meningiomas, although its precise molecular and clinical roles remain poorly understood. METHODS:To comprehensively investigate BAP1-altered meningiomas, we used six meningiomas with known BAP1 alterations as a discovery set. Genome-wide DNA methylation profiling of these samples, along 11,151 reference meningiomas, identified a distinct molecular cluster (n = 42) using unsupervised visualization approaches. These tumors were further characterized by DNA/RNA sequencing, histopathological examination, and a retrospective review of clinical data, compared to reference meningioma cohorts, providing a thorough characterization of this rare tumor subtype. RESULTS:Our integrative analysis revealed BAP1-altered meningiomas as a distinct CNS tumor subtype, characterized by recurrent loss of chromosome 3p21 and driven by various BAP1-inactivating alterations. Although rhabdoid morphology is present in some cases, it is not exclusive and should not be used as a grading criterion. Progression-free survival analysis showed a median of 21 months (95% CI: 12-NA), with a 2-year overall survival rate of 79% (95% CI: 60%-100%), highlighting the aggressive nature of these tumors. Gene expression profiling revealed upregulation of PRC target genes, dysregulated Polycomb signaling, and elevated expression in several cellular and growth factor pathways. CONCLUSIONS:BAP1-altered meningiomas represent a distinct and aggressive CNS tumor subtype associated with PRC dysregulation and recurrent 3p chromosome loss. These findings support the designation "meningioma, BAP1-altered."

AIMS/OBJECTIVE:We investigated key signalling pathways' activity and mutational status of early-stage breast carcinomas with low and intermediate 21-gene recurrence score (RS) to identify molecular features that may predict recurrence. METHODS:This is a retrospective case-control study of 18 patients with recurrent breast carcinoma with low and intermediate 21-gene RS (<25) and control group of 15 non-recurrent breast cancer patients. DNA and mRNA were extracted from tumour tissue. mRNA expression of genes involved in oestrogen receptor (ER), androgen receptor (AR), PI3K and MAPK signalling pathways was measured by real-time quantitative reverse transcription-qPCR (OncoSIGNal G4 test, InnoSIGN). Tumour mutational landscape was assessed by targeted DNA sequencing (Oncomine Precision Assay). RESULTS:mutations, may play a role in the recurrence of early-stage breast cancer with low and intermediate 21-gene RS. Pathway analysis can help to identify high-risk patients in this setting.

Decreased brain levels of coenzyme Q₁₀ (CoQ₁₀), an endogenously synthesized lipophilic antioxidant^1,2, underpin encephalopathy in primary CoQ₁₀ deficiencies^3,4 and are associated with common neurodegenerative diseases and the ageing process^5,6. CoQ₁₀ supplementation does not increase CoQ₁₀ pools in the brain or in other tissues. The recent discovery of the mammalian CoQ₁₀ headgroup synthesis pathway, in which 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL) makes 4-hydroxymandelate (4-HMA) to synthesize the CoQ₁₀ headgroup precursor 4-hydroxybenzoate (4-HB)⁷, offers an opportunity to pharmacologically restore CoQ₁₀ synthesis and mechanistically treat CoQ₁₀ deficiencies. To test whether 4-HMA or 4-HB supplementation promotes CoQ₁₀ headgroup synthesis in vivo, here we administered 4-HMA and 4-HB to Hpdl^-/- mice, which model an ultra-rare, lethal mitochondrial encephalopathy in humans. Both 4-HMA and 4-HB were incorporated into CoQ₉ and CoQ₁₀ in the brains of Hpdl^-/- mice. Oral treatment of Hpdl^-/- pups with 4-HMA or 4-HB enabled 90-100% of Hpdl^-/- mice to live to adulthood. Furthermore, 4-HB treatment stabilized and improved the neurological symptoms of a patient with progressive spasticity due to biallelic HPDL variants. Our work shows that 4-HMA and 4-HB can modify the course of mitochondrial encephalopathy driven by HPDL variants and demonstrates that CoQ₁₀ headgroup intermediates can restore CoQ₁₀ synthesis in vivo.

BACKGROUND:Molecular aberrations have been incorporated into tumour classification guidelines of meningioma. TERT-promoter (TERTp) mutation is associated with worse prognosis and is designated a WHO grade 3 biomarker. However, it remains unclear whether TERTp mutation is context-dependent, with other co-occurring genetic alterations potentially driving its association with prognosis. We sought to characterise the role of TERTp mutation in meningioma and guide TERTp sequencing. METHODS:We identified 1492 patients of all ages who had previously received surgery for meningioma across 14 medical centres in the USA, Canada, and Germany. Patients were eligible if they had post-surgical clinical or radiographical assessment of the resection site, and TERTp status evaluated by Nov 1, 2024. Multi-modal profiling was used to assess TERTp mutation, focal gene alterations-including CDKN2A/B loss-and copy number alterations. An adjusted WHO grade was calculated for TERTp-mutant meningiomas, incorporating all WHO criteria except TERTp status. Kaplan-Meier curves and multivariable Cox proportional hazards models were used to quantify the effect of TERTp mutation on the endpoints of overall survival and recurrence-free survival across adjusted WHO grade and co-occurring molecular alterations. FINDINGS/RESULTS:64 (4·3%) of 1492 meningiomas were TERTp-mutant and 1428 (95·7%) were TERTp-wildtype. Of the TERTp-mutant meningiomas, 33 (51·6%) were from female patients and 31 (48·4%) were from male patients, and the overall median age was 67 years (IQR 60-75). Of the wildtype meningiomas, 965 (67·6%) were from female patients and 463 (32·4%) were from male patients, and the overall median age of the patients was 59 years (IQR 48-70). Data on race was inconsistently reported and thus excluded. The TERTp-mutant patients had a 5-year overall survival (49·4% [95% CI 33·7-72·4]) and 5-year recurrence-free survival (27·6% [95% CI 16·8-45·5]) resembling that of patients with WHO grade 3 TERTp-wildtype tumours (5-year overall survival 32·3% [95% CI 17·2-60·5], p=0·28, 5-year recurrence-free survival 14·3% [5·8-35·2], p=0·28). However, the TERTp-mutant group had heterogenous histological grading and was enriched for aggressive molecular features, with 1p loss present in 44 (77·2%) of 57 profiled tumours and CDKN2A/B loss in 24 (41·4%) of the 58 profiled tumours. Adjusting tumour grade revealed a subset of TERTp-mutant meningiomas that were more molecularly and clinically benign. Among TERTp-mutant tumours, CDKN2A/B loss played a defining role in stratifying tumour behaviour. Multivariable analysis confirmed this, with CDKN2A/B loss being significantly associated with shorter overall survival (HR 3·04 [95% CI 1·67-5·52], p=0·00026) and faster time to recurrence (HR 5·22 [95% CI 3·10-8·79], p<0·0001), while TERTp-mutation did not independently affect overall survival (HR 1·00 [95% CI 0·53-1·87], p=0·99) or recurrence-free survival (1·17 [95% CI 0·75-1·83], p=0·49). Sequencing for TERTp-mutation demonstrated clinical impact only among histologically WHO grade 2 meningiomas. INTERPRETATION/CONCLUSIONS:The indolent behaviour of certain TERTp-mutant meningiomas suggests that TERTp mutation is not sufficient to assign the most aggressive meningioma grade. Instead, TERT sequencing might offer prognostic utility in identifying high-risk cases among WHO grade 2 meningiomas. FUNDING/BACKGROUND:National Institutes of Health, National Institute of Neurological Disorders and Stroke, Friedberg Charitable Foundation, Courtney Meningioma Research Fund, Fleming Meningioma Research Fund, and the Gray Family Foundation.

PURPOSE/OBJECTIVE:) disease. NRG Oncology BN002 (phase I) demonstrated safety and suggested efficacy of ipilimumab (ipi) with nivolumab (nivo) in newly diagnosed glioblastoma, leading to this phase II/III trial. METHODS:) of .15, superior PFS with immunotherapy in phase II would lead to phase III overall survival (OS) testing. Corticosteroids were disallowed when starting immunotherapy. Diagnosis, biomarkers, and PFS were centrally assessed. RESULTS:= .36). CONCLUSION/CONCLUSIONS:glioblastoma versus TMZ. Accrual closed permanently; the trial will not proceed to phase III. No new safety signals were identified. Molecular correlative analyses and survival follow-up are ongoing.