1p Loss Research Articles

EPCO-12. MENINGIOMA COPY NUMBER ANALYSIS IDENTIFIES OPTIMIZED SIZE THRESHOLDS AND CO-OCCURRENCE MODELS FOR INDIVIDUALIZED RISK-STRATIFICATION

Abstract Arm-level copy number alterations (CNAs) are common in meningiomas and are useful for risk-stratification, but there is no consensus regarding the optimal size threshold to define CNAs for prognostic models. Moreover, the prognostic importance of co-occurring CNA pairs and overall CNA burden in meningiomas remains incompletely understood. To address this, CNAs were analyzed in 691 retrospective meningiomas with clinical data and DNA methylation profiles from 3 international institutions. Arm-level CNAs were defined from DNA methylation profiles using iterative size thresholds ranging from 1-99% of each chromosome arm. The area under the curve (AUC) for 5-year local freedom from recurrence (LFFR) or overall survival (OS) was calculated using CNAs across size thresholds, and AUC standard deviation across thresholds was used as a measure of size-dependence. LASSO and Elastic Net Cox regressions were used to generate multi-CNA models for LFFR or OS and to identify prognostic co-occurring CNA pairs. These analyses identified 13 size-dependent prognostic CNAs in meningiomas, including loss of 1p, 3q, 4p, 4q, 6p, 6q, 9p, 10q, 12q, 14q,18q, and 22q, and gain of 1q. Regression models restricted to size-dependent CNAs improved risk stratification within strata of previously published models (Integrated score, Integrated grade), and identified prognostic CNAs not included in previous models, such as 1q gain, 7p loss, and 12q loss. Ontology analysis of genes in focal prognostic CNAs identified dysregulated STAT signaling and decreased expression of interferon-related genes from matched paired RNA sequencing. Models of size-dependent, prognostic, co-occurring CNA pairs identified 1p/22q codeletion, 1q gain/22q loss, and 9p/18q codeletion as significant predictors of LFFR. Increased CNA burden correlated with higher tumor grade and was significantly associated with worse LFFR and OS. In summary, chromosome-specific size thresholds identify prognostic arm-level CNAs and co-occurring CNA pairs in meningiomas. Thus, granular copy number analysis may improve risk stratification models for meningioma.

PATH-04. THE PROGNOSTIC IMPACT OF CDKN2A/B HETEROZYGOUS DELETIONS IN MENINGIOMA- INSIGHTS OF A MULTICENTER ANALYSIS

Abstract BACKGROUND Meningiomas are one of the most common primary brain tumors. Homozygous deletions of CDKN2A/B have reliably been shown to confer an increased risk of progression. However, the role of heterozygous CDKN2A/B deletions remains less clear. MATERIAL AND METHODS DNA methylation data, copy-number information and mutation data were generated on a multicenter cohort of meningiomas for a total of 970 samples. The CDKN2A/B locus was determined manually for each individual case in relation to whole chromosomal or larger segmental losses and gains in the chromosomal profile. Survival probabilities were assessed using the Kaplan–Meier method. RESULTS A total of 970 meningioma cases were identified with 30 homozygous and 114 heterozygous CDKN2A/B deletion cases, 826 cases had balanced CDKN2A/B. Heterozygous CDKN2A/B deletions in general did not confer an increased risk of progression compared to CDKN2A/B balanced cases (p= 0.074). However, segmental heterozygous losses of 9p, encompassing CDKN2A/B, yielded a significantly worse prognosis when compared to meningiomas CDKN2A/B-balanced (p= 0.002) and in contrast to focal heterozygous deletions (p= 0.523). Segmental heterozygous 9p deletions encompassing CDKN2A/B were significantly more frequently associated with a higher amount of copy number variations (CNVs) than focal losses. CONCLUSION Segmental heterozygous CDKN2A/B deletions do confer an increased risk of progression, but focal heterozygous CDKN2A/B deletions do not. The signal for segmental deletions may not be locus-specific but just one representation of the generally instable genome of aggressive meningiomas.

PATH-57. CLINICAL AND MOLECULAR FEATURES OF METHYLATION CLASS DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR

Abstract Genome-wide DNA methylation profiling can help identify rare CNS tumors, including diffuse leptomeningeal glioneuronal tumor (DLGNT). Due to the infrequency of this entity, DLGNT remains poorly understood. To help resolve uncertainty regarding DLGNT, we examined epidemiologic, histologic, and molecular features of 32 (21 newly profiled, 11 publicly available) tumors matching to DLGNT by the DKFZ classifier (v12, score >=0.85). Of these tumors, 25 matched to DLGNT subtype 1 and seven to subtype 2. Subject median age was 12 years in subtype 1 and 22 years in subtype 2 (14.5 overall, range 2-44), with equal numbers of females and males (n=16 each). Spinal cord was the most frequently involved site (n=21), but intracranial lesions were observed (n=6). Leptomeningeal involvement was noted in only four of 11 tumors with available imaging descriptions. In the subset of cases available for histologic review (n=19), oligodendrocyte-like morphology was present in over half (n=11). Microvascular proliferation was uncommon (n=5). Chromosome 1p loss was observed in 29 (91%), and 1p/19q co-deletion was observed in 18 tumors (56%). Gains of 1q and 7q were each observed in 12 (38%), and whole chromosome 7 gain was observed in eight (25%). Loss of 22q was seen in 12 (38%). In the subset of tumors with fusion testing (n=15), KIAA1549::BRAF fusion was present in 12 (80%), and one showed a QKI::RAF1 fusion. Five tumors harbored BRAF mutations, and one harbored a break-point in BRAF without a known fusion partner. In nine subjects with clinical follow-up, there was one death after 3.1 years, and the remaining subjects were alive with a median follow-up of 6.4 years (maximum 19 years). Progression-free survival (PFS) was available for six subjects with a median PFS of 6 years. Presentation of DLGNT without noted leptomeningeal involvement appears common. Further characterization of subject outcomes will help clarify the clinical behavior of DLGNT.

PATH-17. MOLECULAR CLASSIFICATION OF H3K27-WILDTYPE SPINAL CORD GLIOMAS: IMPLICATIONS FOR PROGNOSIS AND THERAPEUTICS

Abstract BACKGROUND H3K27-wildtype spinal cord gliomas (H3WT-SCGs) are rare and heterogeneous tumors that present significant challenges in pathological classification. Traditional histologic diagnoses often do not align well with the underlying molecular characteristics of these tumors. METHODS We characterized 55 cases of H3WT-SCGs using a comprehensive approach that included DNA methylation array, whole-exome sequencing, transcriptome sequencing, proteomics, and single-cell RNA sequencing. Unsupervised clustering analysis was performed to identify distinct molecular subtypes. RESULTS The analysis identified seven distinct spinal cord methylation (SCM) clusters: pilocytic astrocytomas (PA-SPINE), PA-immune enriched (PA-IME), diffuse leptomeningeal glioneuronal tumor (DLGNT), subependymoma (SUBEPN-SPINE), glioblastoma-like (GBM-like), and pleomorphic xanthoastrocytoma-like (PXA-like). There was a 61.8% discordance between SCM classes and traditional histologic diagnoses. The new molecular classification provided a better correlation with molecular features and patient prognosis. BRAF fusions were enriched in PA-SPINE and DLGNT (p = 0.04), and DLGNT showed significant chromosomal alterations, including 1p loss (p = 6.5e-08) and 1q gain (p = 3.6e-05). Notably, we identified two distinct classes of PA-SPINE: adult (older than 18 years) and pediatric. Adult PAs exhibited lower frequencies of BRAF fusions (p = 1.4e-03), shorter tumor lengths (p = 3.7e-04), and longer progression-free survival (p = 0.062). Adult PA neoplastic cells were less differentiated, with higher expression of EGFR and neuronal markers, and lower cytokine and chemokine expression compared to pediatric cases. Single-cell analysis revealed reduced infiltration of monocyte-derived macrophages and proliferative myeloid cells in adult cases. Both PA-IME and PA-pediatric clusters were characterized as immune-hot tumors with high immune cell enrichment. CONCLUSION This study defined clinically relevant molecular classes of H3WT-SCGs that better reflect patient prognosis and molecular characteristics compared to traditional histologic diagnoses. The findings suggest potential therapeutic implications, especially in immune-hot tumors like IME and PA-pediatric, which could benefit from targeted immunotherapies.

BIOM-60. UNDERSTANDING THE INTERSECTION OF RACE, SEX, AND GENOMIC PROFILES IN MENINGIOMA PATHOPHYSIOLOGY

Abstract BACKGROUND While meningioma incidence and outcomes vary by sex and race, the potential underlying molecular differences possibly contributing to these disparities are less understood. Herein, we explore the clinical and genomic characteristics of meningiomas across groups characterized by age and sex. METHODS We analyzed adult patients with primary meningiomas resected from 2012 to 2023. Patients underwent whole exome sequencing, and demographic data were categorized into four groups based on self-reported race and sex, which included White females, White Males, Black females, and Black males. The primary analyses involved comparisons of tumor characteristics, socioeconomic status (median zip code income, % with high school diploma, health insurance status, distance from hospital), genomics, and recurrence rates. RESULTS The study included 509 primary meningiomas. White females exhibited low-risk genomic profiles characterized by driver mutations in KLF4 (+/- TRAF7) (p=0.010) and POLR2A (p=0.045), with no associated CNVs (p=0.410). This benign molecular profile correlated with lower WHO grades (p=0.013) and a lower incidence of recurrence (p=0.0003). Black females had meningiomas enriched with Hedgehog pathway mutations (p=0.0035) and a predilection for the anterior skull base (p=0.034), leading to an increased risk of recurrence (p=0.026). White males showed an aggressive genomic profile, including chromosome 1p loss (p=0.025) and 10q loss (p=0.067), associated with a high risk of recurrence (p=0.0002). Black males had meningiomas with chromosome 14q loss (p=0.0002), the highest incidence of high-grade meningiomas (p=0.048), and a 4.5-fold increased risk of recurrence (OR 4.5 [1.6-11.8]; p=0.006). Black males also experienced the worst progression-free survival (HR 5.5 [2.5-12.4]; p<0.0001), independent of WHO grade, extent of resection, and socioeconomic status. CONCLUSIONS Each demographic has a distinct molecular profile that correlates with clinical behavior in a race and sex-specific manner. Black males are disproportionately affected by biologically aggressive meningiomas, leading to higher tumor grade and recurrence rates.

Angiomyolipomatous Lesions of the Nasal Cavity (Sinonasal Angioleiomyoma with Adipocytic Differentiation): A Multi-Institutional Immunohistochemical and Molecular Study.

Mesenchymal neoplasms composed of vascular, smooth muscle, and adipocytic components are uncommon in the nasal cavity. While angioleiomyoma (AL) is a smooth muscle tumor in the Head & Neck WHO classification, it is considered of pericytic origin in the Skin as well as Soft Tissue and Bone classifications. For nasal AL with an adipocytic component, the terms AL with adipocytic differentiation and angiomyolipoma (AML) have been applied, among others. AML is a type of perivascular epithelioid cell tumor (PEComa), most often arising in the kidney, sometimes associated with the tuberous sclerosis complex (TSC). It is uncertain whether nasal cavity AML and AL are best considered hamartomas or neoplasms, as their genetics are largely unexplored. We performed a multi-institutional retrospective study of nasal cavity mesenchymal lesions. Patient demographics, clinical histories, and histologic and immunohistochemical findings were collected. DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue and analyzed by SNP-based chromosomal microarray, targeted RNA fusion sequencing, and whole-exome sequencing. Fifteen lesions (3-42mm) were identified, predominantly in male (87%) patients with a median age of 60. Patients typically presented with obstructive symptoms, and none had a history of TSC. One AL was a recurrence from six years prior; 11 cases showed no recurrence (median 4.7 years, range: 0.88-12.4). Morphologically, 11 AML contained 30-80% smooth muscle, 10-25% vasculature, and 2-60% adipose tissue, while four AL contained 70-80% smooth muscle and 20-30% vasculature. Other histologic observations included ulceration, thrombosis, inflammation, myxoid change, senescent nuclei, and extramedullary hematopoiesis; no well-developed epithelioid cell morphology was identified. Immunohistochemically, all cases were positive for smooth muscle markers (actin, desmin, and/or caldesmon) and negative for melanocytic markers. Molecular analysis revealed loss of 3p and 11q in a single AML. No other known pathogenic copy number or molecular alterations were seen, including in TSC1/2, TFE3, or NOTCH2. Nasal cavity AML lacks morphologic, immunophenotypic, and genetic features of PEComa family AML. The significant histologic overlap between nasal AML and AL without distinguishing molecular features in either entity suggests "sinonasal angioleiomyoma with adipocytic differentiation" may be the most appropriate terminology for hybrid vascular and smooth muscle lesions containing adipocytic components.

Segmental chromosome aberrations as a prognostic factor of neuroblastoma: a meta-analysis and systematic review.

Segmental chromosome aberrations, defined as presence of aberrations, deletion, or imbalance in the chromosomal arms, have long been considered as a predictor of poor prognosis of patients with neuroblastoma. The objective of this meta-analysis is to quantitively analyze the hazard ratios (HRs) of different whole or segmental chromosome aberrations for overall survival (OS) rate or event-free survival (EFS) rate of patients with neuroblastoma. Relevant studies about chromosome, neuroblastoma, predictor, prognosis, and survival published from the inception to April 2023 in the databases of PubMed, Embase, and Web of Science were searched, screened, and reviewed. The risk of bias of included articles was assessed using the Quality In Prognosis Studies tool. Basic characteristics, HRs of long term (>3 years) EFS and OS with 95% confidence intervals (CIs) of included articles were extracted. A random effects model of DerSimonian-Laird was used to analyze the extracted HRs. For studies that did not report HRs, narrative synthesis was used for summarization. There were 34 (including 14,356 patients) in 844 searched studies finally included for narrative and quantitative analysis. There were 24 articles rated as low risk of bias and 10 articles rated as moderate. Although the results were inconsistent, the pooled effect of HR for 1p loss was 4.46 (1.88-10.59) for EFS and 2.29 (1.26-4.15) for OS; the pooled effect of HR for 17q gain was 4.81 (3.29-7.04) for EFS and 3.98 (2.11-7.54) for OS; the pooled effect of HR for 11q loss was 2.54 (2.32-3.73) for OS. Results of 1p36 loss, 1p22 loss, 11q23 loss, 11q13-q14 gain, 1q gain, 1q22 gain, 2p gain, 3p loss, 4p loss, 14q loss, 14q32 loss, and other segmental chromosome aberrations were also summarized. 1p loss, 11q loss, and 17q gain were identified as significant independent predictors for long-term OS and EFS of patients with neuroblastoma.

Angiomyolipomatous Lesions of the Nasal Cavity (Sinonasal Angioleiomyoma with Adipocytic Differentiation): A Multi-Institutional Immunohistochemical and Molecular Study.

Mesenchymal neoplasms composed of vascular, smooth muscle, and adipocytic components are uncommon in the nasal cavity. While angioleiomyoma (AL) is a smooth muscle tumor in the Head & Neck WHO classification, it is considered of pericytic origin in the Skin as well as Soft Tissue and Bone classifications. For nasal AL with an adipocytic component, the terms AL with adipocytic differentiation and angiomyolipoma (AML) have been applied, among others. AML is a type of perivascular epithelioid cell tumor (PEComa), most often arising in the kidney, sometimes associated with the tuberous sclerosis complex (TSC). It is uncertain whether nasal cavity AML and AL are best considered hamartomas or neoplasms, as their genetics are largely unexplored. We performed a multi-institutional retrospective study of nasal cavity mesenchymal lesions. Patient demographics, clinical histories, and histologic and immunohistochemical findings were collected. DNA and RNA were extracted from formalin-fixed, paraffin-embedded tissue and analyzed by SNP-based chromosomal microarray, targeted RNA fusion sequencing, and whole-exome sequencing. Fifteen lesions (3 to 42 mm) were identified predominantly in male (87%) patients with a median age of 60. Patients typically presented with obstructive symptoms, and none had a history of TSC. One AL was a recurrence from six years prior; 11 cases showed no recurrence (median 4.7 years, range: 0.88-12.4). Morphologically, 11 AMLs contained 30-80% smooth muscle, 10-25% vasculature, and 2-60% adipose tissue, while four ALs contained 70-80% smooth muscle and 20-30% vasculature. Other histologic observations included surface ulceration, vascular thrombosis, chronic inflammation, and myxoid change; no well-developed epithelioid cell morphology was identified. Immunohistochemically, all cases were positive for smooth muscle markers (actin and/or desmin) and negative for melanocytic markers. Molecular analysis revealed loss of 3p and 11q in a single AML. No other known pathogenic copy number or molecular alterations were seen, including in TSC1/2, TFE3, or NOTCH2. Nasal cavity AML lacks morphologic, immunophenotypic, and genetic features of PEComa family AMLs. The significant histologic overlap between nasal AML and AL without distinguishing molecular features in either entity suggests "sinonasal angioleiomyoma with adipocytic differentiation" may be the most appropriate terminology for hybrid vascular and smooth muscle lesions containing adipocytic components.

Spatial Distribution of Meningiomas: A Magnetic Resonance Image Atlas.

The size and anatomic location of meningiomas have been shown to correlate with distinct clinical manifestations, histopathological subtypes, and surgical risk. However, meningioma anatomic origin sites can be obscured in large tumors and those crossing compartments. We therefore sought to apply unbiased lesion mapping to localize intracranial meningioma distributions and their association with biology and grade. MRI scans, World Health Organization (WHO) grade, and a molecularly Integrated Grade (IG) derived from cytogenetics were analyzed from adult patients with intracranial meningiomas. Semi-automated tumor segmentation was performed on T1-weighted contrast-enhanced MRI. We used the voxel-based lesion mapping technique to generate a meningioma atlas, mapping spatial frequency and correlating with tumor grades. Of 881 patients with meningioma (median age: 57 years, 68.8% female), 589 were WHO grade 1 (66.8%), 265 WHO grade 2 (30.1%), and 27 WHO grade 3 (3.1%) with a median tumor volume of 14.6 cm3. After molecular reclassification, 585 were IG-1 (66.4%), 160 IG-2 (18.2%), and 136 IG-3 (15.4%). Benign tumors were concentrated in and around the midline anterior skull base while malignant meningiomas were enriched in the falcine/parasagittal region and the sphenoid wing, similar to the distribution when stratified by chromosome 1p loss. Meningiomas exhibited sharper spatial clustering when stratified by the molecular IG than by WHO grade. WHO grade 2 meningiomas divided equally across IG 1-3, with corresponding partition of spatial distribution in the midline anterior skull base (in WHO grade 2, IG-1) and falcine/parasagittal and sphenoid regions (WHO grade 2, IG-3). Meningioma volumes significantly varied across age, sex, and WHO/IG grades. We demonstrate the utility of voxel-based lesion mapping for intracranial tumors, characterizing distinct meningioma distribution patterns across histopathological and molecularly defined grades. Molecular grading associated with sharper tumor spatial clusters, supporting a phenotype-genotype association in meningiomas.

16 Linking VHL and SETD2 in a common oncogenic pathway that converges on the mitoticspindle

Abstract Background Loss of chromosome 3p is a landmark event in clear cell renal cell carcinoma (ccRCC) that results in mono-allelic loss of VHL (von Hippel Lindau) and SETD2 (Set-domain containing 2) (and other tumor suppressors co-located on 3p). Second hits in VHL inactivate this key tumor suppressor initiating tumor progression. SETD2, a histone methyltransferase, was previously shown to have a dual function in methylating both histones and microtubules, thereby contributing to both the histone and tubulin codes. Methylation by SETD2 on microtubules occurs at the mitotic spindle and is essential for normal mitosis and cytokinesis, with loss of SETD2 acting as a strong driver of apoptosis. This raises a conundrum of how cancer cells survive early mono-allelic loss of SETD2, escaping cell death. Methods Using biochemical kinase assays and mass spectrometry we have identified SETD2 as a substrate for AURKA. In addition, we have used immunoblotting and immunofluorescence assays to probe phosphorylation on SETD2 and the impact of phosphorylation of SETD2 both on its chromatin and cytoskeleton targets. Results We have identified the mitotic kinase, Aurora kinase A (AURKA), as a regulator of SETD2. Our data uncover SETD2 as a unique substrate for phosphorylation by AURKA, with mass spectrometry identifying serine 2080 (S2080) as the site of phosphorylation on SETD2. We found phosphorylation of SETD2 by AURKA at S2080 contributes to its methyltransferase (i.e. enzymatic) activity on microtubules but does not impact chromatin methylation on H3K36 which remains unaltered. We show that VHL regulates SETD2 via AURKA, and loss of phosphorylation on SETD2 results in mitotic defects and genomic instability. Importantly, we demonstrate that inhibition of AURKA is synthetic lethal in the setting of VHL and SETD2 deficiency. Conclusions AURKA expression levels are high in VHL-null cells resulting from an inability of VHL to target AURKA for degradation and our data now highlight a direct link between VHL and SETD2, two tumor suppressors believed to independently drive RCC pathogenesis. In summary, our data reveal a tumor-specific vulnerability linked to mitotic fragility that can be precisely targeted to ultimately drive mitotic catastrophe. DOD CDMRP Funding: yes

Copy Number Profiling Implicates Thin High-Grade Squamous Intraepithelial Lesions as a True Precursor of Cervical Human Papillomavirus-Induced Squamous Cell Cancer

Full-thickness high-grade squamous intraepithelial lesions (HSIL) are precursors of invasive cervical squamous cell carcinoma (SCC). The World Health Organization and Lower Anogenital Squamous Terminology Standardization Project for human papilloma virus (HPV)–associated lesions divide full-thickness HSIL of the cervix into thin HSIL with thickness of 1 to 9 cell layers and the typical full-thickness HSIL of >10 cell layers. Although HPV oncogene transcripts and p16ink4a overexpression, as markers of transforming HPV infection, are detectable in thin HSIL, the biological significance of thin HSIL in cervical carcinogenesis remains poorly understood. To further characterize thin HSIL, we performed a comparative study of chromosomal copy number variations (CNV), an analysis of dysregulated genes present in the segments with CNV, and a generalized genetic complexity calculation for 31 thin HSIL, 31 thick HSIL, 24 microinvasive SCC (pT1a SCC), and 22 highly invasive SCC samples. Thin HSIL share various CNV and specific dysregulated gene pathways with thick HSIL and invasive SCC. Thin HSIL exhibited an average CNV of 11.6% compared with 14.1% for thick HSIL, 15.5% for pT1a SCC, and 26.6% for highly invasive SCC. The CNV included gains at 1q and 3q (40% and 43%, respectively), partial loss of 3p, and loss of chromosomes 11 (18%), 16 (50%), 20 (35%), and 22 (40%). Pathways affected solely in thin HSIL were those enhancing immune evasion and primarily involved the (interleukin) IL6, IL21, and IL23 genes. ILs are transiently upregulated in response to infection and play a crucial role in mounting antitumor T-cell activity. Deregulation reflects an attempt by the HPV to evade the initial immune response of the host. The primary pathways shared by thick HSIL and invasive SCC were interactions between lymphoid and nonlymphoid cells, NOTCH2 signaling, tight junction interactions (primarily of the claudin family), and FGR2 alternative splicing. Our results show that thin HSIL carry similar genetic changes as thick HSIL and SCC, indicating that thin HSIL are true precursor lesions that can progress to thick HSIL and SCC.

Protein and mRNA Expression in Uveal Melanoma Cell Lines Are Related to GNA and BAP1 Mutation Status.

Cell lines are being used in preclinical uveal melanoma (UM) research. Because not all cell lines harbor typical GNAQ or GNA11 hotspot mutations, we aimed at better classifying them and determining whether we could find genetic causes to explain the protein and mRNA expression profiles of the cell lines. We studied protein and mRNA expression of 14 UM cell lines and determined the presence of single nucleotide variants and small insertions and deletions with next-generation sequencing and copy number alterations with a single nucleotide polymorphism array. The lists of differentially expressed proteins and genes were merged, and shared lists were created, keeping only terms with concordant mRNA and protein expression. Enrichment analyses were performed on the shared lists. Cell lines Mel285 and Mel290 are separate from GNA-mutated cell lines and show downregulation of melanosome-related markers. Both lack typical UM mutations but each harbors four putatively deleterious variants in CTNNB1, PPP1R10, LIMCH1, and APC in Mel285 and ARID1A, PPP1R10, SPG11, and RNF43 in Mel290. The upregulated terms in Mel285 and Mel290 did not point to a convincing alternative origin. Mel285 shows loss of chromosomes 1p, 3p, partial 3q, 6, and partial 8p, whereas Mel290 shows loss of 1p and 6. Expression in the other 12 cell lines was related to BAP1 expression. Although Mel285 and Mel290 have copy number alterations that fit UM, multi-omics analyses show that they belong to a separate group compared to the other analyzed UM cell lines. Therefore, they may not be representative models to test potential therapeutic targets for UM.

OTHR-25. ANAPLASTIC TRANSFORMATION OF DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMORS: TWO PEDIATRIC CASES

Abstract BACKGROUND Diffuse Leptomeningeal Glioneuronal Tumors (DLGT) typically have a clinical course with slow progression; however, there have been cases of anaplastic transformation. We describe the clinical presentation, radiographic features, and molecular characteristics in 2 pediatric DLGT cases that underwent aggressive transformation. METHODS Patient 1: A 20 y/o male with DLGT with BRAF-KIAA1549 fusion and loss of 1p who was first diagnosed at 2.5 y/o. Over the course of 17 years, he was treated with numerous therapies due to multiple progressions, including temozolomide, carboplatin/vincristine, selumetinib, and vinblastine. At age 17, he developed significant growth of a posterior fossa mass with solid and cystic characteristics. Resection revealed new anaplastic pathologic features. Patient 2: He was diagnosed with DLGT at age 2 y/o. Imaging demonstrated intramedullary spinal nodules at T4 and T5 with abnormal thickening and enhancement over the cerebral leptomeningeal space. A biopsy revealed DLGT with a BRAF-KIAA1549 fusion, 1p loss and Ki67 5%. He received multiple therapies including carboplatin/vincristine, vinblastine, and selumetinib. RESULTS Patient 1: Molecular characterization revealed new ATRX mutation, and CDKN2A/B homozygous deletion. Over the following 2 years, he had multiple treatments, progressions, and surgeries. Final pathology revealed significant anaplasia with Ki67 90%, ATRX loss, and mutation of PPM1D and ARID1A. He rapidly declined. Patient 2: Approximately 2.5 years after diagnosis, he developed rapid progression in the 4th and lateral ventricles. Pathology confirmed recurrent DLGT, but with high-grade transformation, H3K27M (-), H3k27m3 loss, ATRX (+), Ki67 40%. He was treated with focal irradiation followed by temozolomide/CCNU. CONCLUSION In rare cases, DLGT can undergo anaplastic transformation. Once this occurs, it behaves clinically like a high-grade glioma. Understanding the molecular underpinnings and prognostic features that lead to this transformation are important for future study.

DEI-02. DIRECT PATIENT OUTREACH IN PEDIATRIC NEURO-ONCOLOGY TO IMPROVE ACCESS TO GENOMIC PROFILING – THE MAKE-AN-IMPACT PROGRAM

Abstract BACKGROUND Genomic profiling and precision medicine have revolutionized cancer care but are not accessible to all due to high costs, insurance issues, limited access to testing facilities or qualified personnel. We established a program providing free clinical genomic testing worldwide (Make-An-IMPACT) and report findings from the pediatric CNS tumor cohort. METHODS Following a referral from: (1) a physician, (2) a disease-specific advocacy group, or (3) submission of contact information through program website, patients were enrolled on an IRB-approved protocol (NCT01775072). CSF cell free DNA (cfDNA) and matched normal samples were analyzed using MSK-IMPACTTM, a New York State Department of Health authorized hybridization capture-based next-generation panel DNA sequencing clinical assay. If a driver mutation wasn’t reported, raw genomic data was reviewed. OncoKBTM was used to determine the level of evidence supporting the clinical actionability of genomic aberrations. RESULTS 24 patients (median age 7.5; range 0.1-20 years) were enrolled; 9 (38%) from 5 non-US countries and 6 (25%) from non-NYC US sites. African American and Hispanics constituted 8% and 13% of the cohort. Potentially actionable alterations were identified in 4/24 (17%) cases; 2/24 (8%) subsequently received matched therapy. In unbiopsied or non-diagnostic cases, a disease defining alteration was identified in 8/15 (53.3%). BRAF-KIAA1549 fusions were identified in 2/3 DLGNT patients and 1p loss in all 3. Both patients with BRAF fused DLGNT started targeted therapy with Trametinib, one of whom achieved a durable treatment response for 2.5 years. 3/8 patients with unbiopsied brainstem lesions had a H3.3K27M mutation, while 2 had non-H3.3K27M mutations detected (1 SMARCB1 deletion, 1 IDHR172G mutation) leading to treatment regimen changes. CONCLUSIONS This study captured a broad range of pediatric CNS malignancies with a diverse patient representation across geographic regions. CSF cfDNA profiling provided important diagnostic and therapeutic information reported in real time to patients and local physicians.

MDB-63. AN INTERNATIONAL META-ANALYSIS OF SHH MEDULLOBLASTOMA SUBTYPES DEFINES A CLINICALLY SIGNIFICANT HIGH-RISK VARIANT OF SHH-SUBTYPE 3

Abstract BACKGROUND The distinction of MBSHH into four methylation-dependent subgroups was recognized by the WHO in 2021. However, these subgroups have not previously been defined by international experimental consensus and their clinico-molecular features and behavior have not formally been investigated in large cohorts. We aimed to robustly identify SHH subgroups through analysis of MBSHH with DNA-methylation profiling. METHODS A cohort of 683 MBSHH, confidently classified using the Heidelberg classifier v12.5, was assembled for analysis from multiple international studies. To define subgroups, we applied consensus sampling-based clustering approaches to tumor methylomes, including assessment of confidence in class-definition and inter-technique concordance. The clinico-molecular features of consensus subgroups were investigated. RESULTS Lowest complexity analysis supported the division of MBSHH into the 4 WHO-subtypes. SHH-1 and SHH-2 were associated with lower age and infrequent mutations of PTCH1/SUFU; SHH-2 was distinguished from SHH-1 by enriched MBEN histology, absence of chromosome 2 gain, less frequent metastasis and more favorable overall-survival. SHH-4 presented in older children and adults (3-57; median 24 years), and was primarily defined by mutations in U1-snRNA (67/72). Consensus analyses supported the division of SHH-subgroup-3 into three. Importantly, the SHH-3C subtype was associated with a coalescence of high-risk features (LCA histology, TP53mut, MYCNamp/GLI2amp) alongside 3p, 10q and 17p loss, and had dismal survival. The remaining SHH-3A/B subtypes were characterized by 9q loss, frequent focal amplifications of TERT and PPM1D, and equivalent better survival. CONCLUSIONS This study affirms the distinction of MBSHH into 4 major subgroups. For infant disease, SHH-2 is a favorable-risk marker. For childhood-MBSHH, the SHH-3C subtype provides a molecular definition of high-risk that subsumes other previously-defined high-risk disease markers (LCA, MYCNamp, TP53mut) into a unified high-risk disease group. The other SHH-3 subtypes behave similarly and are favorable-risk. These subtypes have potential to enhance molecularly-guided risk-stratification in routine diagnostics, to improve patient outcomes and quality-of-life.

Diagnosis and genetic characterization of CNS metastases in lung cancer via shallow whole-genome sequencing of CSF cell-free DNA.

e14013 Background: The genetic characterization of CNS metastases in lung cancer is hindered by the limited sampling opportunities and genetic heterogeneity between intracranial and extracranial lesions. To address these challenges, this study uses cerebrospinal fluid (CSF) and blood samples, to detect and characterize CNS metastasis variants through shallow whole-genome sequencing (sWGS). Methods: From Dec. 2021 to Aug. 2023, 57 lung cancer patients were recruited, comprising 29 with leptomeningeal metastases (LMs) and 28 with only parenchymal brain metastases (PMs). A total of 66 blood and 112 CSF samples were collected for cfDNA sWGS to analyze chromosomal arm CNVs, focal amplifications (CN > 5) and SNV/InDels. Cytology was also conducted on 95 of these CSF samples. Variant differences between PMs and extracranial metastases (EMs) were analyzed using the MSK-MET database. Results: CSF cfDNA concentration was significantly lower than that in plasma (p < 0.0001), with 41.7% of samples unquantifiable, yet all samples successfully completed sWGS. The cfDNA tumor fraction in CSF was significantly higher than in paired plasma (p < 0.0001), and most arm CNVs and gene amplifications exclusively detected in CSF. Cytology, as the gold standard for LM diagnosis, had a sensitivity of 0.76. The sensitivity for LM diagnosis based on aneuploidy and genomic amplification fraction (GAF) were 0.51 and 0.80, respectively, without compromising specificity. Gains in 1p, 7p, 11q, 16p, and 17q, as well as losses in 9p and 9q, were significantly more frequent in PMs compared to EMs and further increased in the LMs (p < 0.05). Notably, losses in 1p, 4p, 5q, and 11p, significantly more frequent in PMs than in extracranial metastases, but showed a significant increase in gain frequency in LMs (p < 0.05). In the LM group, the frequency of EGFR L858R and amplification of nearly the entire 7p where the EGFR located, were significantly increased, whereas KRAS G12D frequency significantly decreased (p < 0.05). Conclusions: The characteristics of extremely low concentration but high tumor fraction of CSF cfDNA make it suitable for sWGS, enhancing LM diagnostic sensitivity without compromising specificity, and identifying molecular characteristics of CNS metastasis. These molecular features require further validation in larger prospective clinical cohorts, offering potential for personalized diagnosis and treatment of CNS metastases.

Paired Primary and Recurrent Rhabdoid Meningiomas: Cytogenetic Alterations, BAP1 Gene Expression Profile and Patient Outcome.

Rhabdoid meningiomas (RM) are a rare meningioma subtype with a heterogeneous clinical course which is more frequently associated with recurrence, even among tumors undergoing-complete surgical removal. Here, we retrospectively analyzed the clinical-histopathological and cytogenetic features of 29 tumors, from patients with recurrent (seven primary and 14 recurrent tumors) vs. non-recurrent RM (n = 8). Recurrent RM showed one (29%), two (29%) or three (42%) recurrences. BAP1 loss of expression was found in one third of all RM at diagnosis and increased to 100% in subsequent tumor recurrences. Despite both recurrent and non-recurrent RM shared chromosome 22 losses, non-recurrent tumors more frequently displayed extensive losses of chromosome 19p (62%) and/or 19q (50%), together with gains of chromosomes 20 and 21 (38%, respectively), whereas recurrent RM (at diagnosis) displayed more complex genotypic profiles with extensive losses of chromosomes 1p, 14q, 18p, 18q (67% each) and 21p (50%), together with focal gains at chromosome 17q22 (67%). Compared to paired primary tumors, recurrent RM samples revealed additional losses at chromosomes 16q and 19p (50% each), together with gains at chromosomes 1q and 17q in most recurrent tumors (67%, each). All deceased recurrent RM patients corresponded to women with chromosome 17q gains, although no statistical significant differences were found vs. the other RM patients.

Abstract 7419: Seq2Karyotype (S2K): A method for deconvoluting heterogeneity of copy number alterations using single-sample whole-genome sequencing data

Abstract Broad copy number alterations (CNAs) at chromosomal band resolution have been used as cancer diagnostic and prognostic biomarkers for decades. These events have been characterized by cytogenetic imaging, an approach which is powerful for assessing heterogeneity but limited for locus fine mapping. While CNA detection by next-generation sequencing has become a standard analysis, existing methods rarely model CNA heterogeneity in bulk tumor samples and often require a paired normal sample for control. To overcome these limitations, we developed Seq2Karyotype (S2K, https://github.com/chenlab-sj/Seq2Karyotype), a new algorithm for in-silico karyotyping using single-sample whole-genome sequencing (WGS) data. S2K performs joint modeling of read-depth and allelic imbalance (AI) of high-quality heterozygous SNPs to identify reference diploid regions, followed by modeling and segmenting the deviation from the reference. Empirical coverage and AI of segmented regions are fitted to models of single and admixed CNAs to estimate clonality. The final karyotyping considers both the model fitness and minimization of evolution steps.To evaluate S2K’s performance, we analyzed two cell lines commonly used for benchmark test: COLO829 (melanoma) and HCT1395 (breast cancer); both had single-cell (sc) WGS for validation. For COLO829, S2K replicated the four populations detected by scWGS but derived a different clonality estimate. The predominant clone, defined by loss of 1p, 10p, and chr18, was estimated to have 67% cellular fraction (CF) by bulk sample analysis of S2K in contrast to the 10% CF by scWGS analysis. In HCT1395, S2K identified three new CNAs present in >50% of the cells of the matching germline sample which were subsequently validated by FISH, karyotyping and SKY mapping.To demonstrate S2K’s utility, we analyzed three diverse data sets: 17 neuroblastoma cell lines, 24 pediatric AML samples with karyotyping data, and two blood samples from children with myelodysplastic syndromes (MDS) known to harbor mosaic uniparental disomy (UPD). CNA-based intra-tumor heterogeneity was detected in 88% (15/17) of the neuroblastoma cell lines, comprised of 2-4 distinct populations with diverse ranges of CF (~10%-90%) and varying CNA patterns (e.g. admixture of tetraploid and diploid cells), which were validated by cytogenetics or scWGS. In the patient AML samples, S2K detected >95% of the previously reported cytogenetic events and 30% of additional copy-neutral loss-of-heterozygosity events. The mosaic UPD events in MDS patients were detected with projected clonality of 60% and 25%, respectively. These results not only demonstrate the accuracy of in-silico karyotyping performed by S2K but also reveal the dynamic intra-tumor heterogeneity in cancer cell lines, which may impact the design and interpretation of future experiments using these cell lines. Citation Format: Limeng Pu, Karol Szlachta, Virginia Valentine, Xiaolong Chen, Jian Wang, Dennis Kennetz, Daniel Putnam, Sivaraman Natarajan, Li Dong, Thomas Look, Marcin Wlodarski, Lu Wang, Steven Burden, John Easton, Xiang Chen, Jinghui Zhang. Seq2Karyotype (S2K): A method for deconvoluting heterogeneity of copy number alterations using single-sample whole-genome sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7419.

Abstract 5049: Detecting chromosomal copy number variations and point mutations in Glioma using a single assay; sparing tissue while significantly reducing testing time and cost

Abstract We aimed to develop a single streamlined assay to detect all Glioma biomarkers. The current diagnosis of Gliomas incorporates molecular biomarkers that include whole chromosomal copy number variations (CNVs) and single nucleotide variations (SNVs). Testing CNVs and SNVs are traditionally done using different large platforms and are performed in large genomic facilities that have the suitable infrastructure. This creates delays in testing, where samples undergo lengthy referrals and the tissue is often split between institutions, which also adds substantially to the costs. We have previously used Nanopore sequencing to develop an assay to detect IDH SNVs from formalin-fixed paraffin-embedded (FFPE) tissue. Nanopore sequencing platform uses small inexpensive tools and can test for both CNVs and SNVs. However, combining both targets efficiently in FFPE tissue remains a challenge. While nanopore whole genome sequencing can detect CNVs, it does not allow for enough coverage to accurately detect small SNVs. Also, raw FFPE DNA is not compatible with the nanopore technology. To fix both challenges we used a PCR based approach that allows enrichment of the SNV targets, as well as creates new nanopore compatible DNA copies. To counteract the bias that PCR creates with uneven amplification, we used a SNP based method to call chromosomal gains and losses. Statistical analysis of probability showed that targeting 15 heterozygous SNPs per chr arm is sufficient for accurate CNV detection (error rate <0.1%). A total of 57 amplicons targeting CNVs on Chr 1,7, 10, 19, and EGFR gene; and IDH1 and IDH2 SNVs were pooled together in one assay and tested on 4 Glioma samples. Variant calling was performed with a custom Python script. All CNV targets were accurately detected on all samples. Losses in chr 1p, 19q and chr 10 were evident with a loss of heterozygosity (LOH) pattern on the SNP analysis. The gain in chromosome 7 was also observed as an allele gain 2:1 SNP pattern on the corresponding samples. The SNV statuses of these samples were also accurately detected when compared to reference results. Finally, the SNV and CNV calling analysis were streamlined using custom shell script, further reducing total turnaround time (including sequencing and data analysis) to less than a day for the entire batch of samples. Initial cost analysis shows <50% of the traditional testing costs for the assay. This work is the first to develop a streamlined single test to detect CNVs and SNVs in Gliomas using FFPE DNA on the nanopore sequencing platform. The shorter testing time, streamlined workflow, and low assay/capital cost could positively influence the care of brain cancer patients. Notably, nanopore-based sequencing tools can also be implemented in other types of tumor diagnosis, especially by smaller labs, helping to overcome many of the existing diagnostic challenges. Citation Format: Mashiat L. Mimosa, Jared T. Simpson, Karel Boissinot, Mora Tiab, Ramzi Fattouh, Rola M. Saleeb. Detecting chromosomal copy number variations and point mutations in Glioma using a single assay; sparing tissue while significantly reducing testing time and cost [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5049.

Abstract 2446: Genomic landscape of longitudinally collected preinvasive airway lesions

Abstract Lung carcinoma in situ (CIS) lesions, as precursors to squamous cell carcinoma, exhibit microscopic uniformity while displaying diverse clinical trajectories. Half of these lesions progress to invasive cancer, while the remaining half either regress or remain indolent. Previous genomic profiling efforts have elucidated progression-specific genomic changes against a backdrop of inherent heterogeneity and notable chromosomal instability signatures. Here, we present a novel approach by analyzing longitudinally collected samples of preinvasive airway lesions, encompassing both progressive and indolent trajectories. Our findings reveal that genomic integrity and cell cycle dynamics constitute key distinctions between progressive and indolent lesions. Notably, a decrease in clonal diversity and an inverse correlation between TCRA-Tcell fraction and genomic instability are observed as lesions approach invasion. Somatic mutations in cancer driver genes BRAC2, CSMD3, and MUC16 are found significantly associated with lesion progression. Furthermore, progressive lesions exhibit a higher frequency of copy number changes compared to indolent lesions. Copy number loss in 1p, 3p, 9p, and 17p are found to be significantly associated with lesion progression. Phylogenetic analysis further confirms positive selection for copy number loss in these specific genomic regions, providing insights into the evolutionary trajectory of alterations in progressive lesions. The comprehensive understanding derived from these genomic analyses is anticipated to advance early detection strategies, mitigate overtreatment, and facilitate the development of preventative therapies targeting early clonal events in lung cancer. Citation Format: Ahmed Alhendi, Lukas Kalinke, Adam Pennycuick, Kate Davies, Pascal Durrenberger, Kate Gowers, Kristiana Grigoriadis, Nicholas McGranahan, Sam Janes. Genomic landscape of longitudinally collected preinvasive airway lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2446.