Chromosome 22q11 Research Articles

RAD21 promotes oncogenesis and lethal progression of prostate cancer

Higher levels of aneuploidy, characterized by imbalanced chromosome numbers, are associated with lethal progression in prostate cancer. However, how aneuploidy contributes to prostate cancer aggressiveness remains poorly understood. In this study, we assessed in patients which genes on chromosome 8q, one of the most frequently gained chromosome arms in prostate tumors, were most strongly associated with long-term risk of cancer progression to metastases and death from prostate cancer (lethal disease) in 403 patients and found the strongest candidate was cohesin subunit gene, RAD21, with an odds ratio of 3.7 (95% CI 1.8, 7.6) comparing the highest vs. lowest tertiles of mRNA expression and adjusting for overall aneuploidy burden and Gleason score, both strong prognostic factors in primary prostate cancer. Studying prostate cancer driven by the TMPRSS2-ERG oncogenic fusion, found in about half of all prostate tumors, we found that increased RAD21 alleviated toxic oncogenic stress and DNA damage caused by oncogene expression. Data from both organoids and patients indicate that increased RAD21 thereby enables aggressive tumors to sustain tumor proliferation, and more broadly suggests one path through which tumors benefit from aneuploidy.

Use of Mitotic Activity and the Size of Any Dedifferentiated Component for Risk Assessment in MDM2-Amplified Liposarcoma.

The characteristic molecular signature for both atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma is amplified sequences derived from chromosome 12q13-15, including MDM2 proto-oncogene (MDM2). As the progression of atypical lipomatous tumor/well-differentiated liposarcoma to the more aggressive dedifferentiated liposarcoma has the potential to adversely affect patient outcomes, the extent of the latter component might be important to evaluate. To investigate the correlation between clinicopathologic characteristics, including tumor size, modified Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC) grade, molecular data, and outcomes in 123 surgically resected MDM2-amplified liposarcomas. Pathology reports and clinical records were reviewed. A log-rank test was used to compare the survival trends, and univariate logistic regression was performed to identify variables associated with adverse events (distant metastasis and/or death), from which the P value was derived to construct a multivariate regression model. In univariate analysis, the largest single dimension of the dedifferentiated component, the percentage of cells with gain of chromosome 12, mitotic count, and the presence of modified FNCLLC grade 3 were associated with adverse events. In multivariate analysis, the largest single dimension of the dedifferentiated component (odds ratio: 1.169; 95% CI: 1.053, 1.299; P = .003), and a higher mitotic count (odds ratio: 1.133; 95% CI: 1.037, 1.237; P = .006) were correlated with adverse events. There was no statistically significant association between current local recurrence status, overall largest tumor dimension, overall tumor volume, MDM2 copy number, or MDM2 to chromosome 12 centromere probe ratio and adverse outcomes. Staging dedifferentiated liposarcoma based on the size of the dedifferentiated component better predicts the outcome.

Short communication: catechol-O-methyltransferase allelic variation in relation to psychological and hormonal indices of stress in children and adolescents with chromosome 22q11.2 deletion syndrome (22q11.2DS).

Chromosome 22q11.2 deletion syndrome (22q11.2DS) is a developmental disorder with high rates of anxiety and psychosis. Catechol-O-methyltransferase (COMT) regulates epinephrine (E), norepinephrine (NE), and dopamine (DA) and is implicated in both anxiety and psychotic disorders. The aim of this study was to determine how COMT variation relates to psychological anxiety and associated stress physiology responsiveness to better understand symptom heterogeneity in people with 22q11.2DS. We examined COMT allelic variation in relation to anxiety and hypothalamic-pituitary-adrenocortical (HPA) and sympathetic-adrenomedullary (SAM) hormonal stress indicators in 30 children and adolescents with 22q11.2DS. Contrary to expectation, individuals with the higher activity COMTval allele had higher anxiety levels versus those with the low activity (COMTmet) allele (p = 0.021; Glass' Δ = 0.69). Anxiety was not correlated with salivary cortisol (CORT) or alpha-amylase (sAA) in either group. Groups did not differ in CORT levels (p = 0.58), but the COMTmet group had higher sAA (p = 0.026; Glass' Δ = 0.67, uncorrected) suggesting greater SAM reactivity but not HPA activity. This suggests that COMT allelic variation may contribute to differences in acute SAM but not slower HPA stress reactivity in those with 22q11.2DS.

Chronic myeloid leukemia: 2025 update on diagnosis, therapy, and monitoring.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm with an annual incidence of two cases/100 000. It accounts for approximately 15% of newly diagnosed cases of leukemia in adults. CML is characterized by a balanced genetic translocation, t(9;22) (q34;q11.2), involving a fusion of the Abelson murine leukemia (ABL1) gene from chromosome 9q34 with the breakpoint cluster region (BCR) gene on chromosome 22q11.2. This rearrangement is known as the Philadelphia chromosome. The molecular consequence of this translocation is the generation of a BCR::ABL1 fusion oncogene, which in turn translates into a BCR::ABL1 oncoprotein. Four tyrosine kinase inhibitors (TKIs), imatinib, dasatinib, bosutinib, and nilotinib, are approved by the United States Food and Drug Administration (FDA) for first-line treatment of newly diagnosed CML in the chronic phase (CML-CP). Clinical trials with second and third-generation TKIs in frontline CML-CP therapy reported significantly deeper and faster responses but had no impact on survival prolongation, likely because of their potent efficacy and the availability of effective TKIs salvage therapies for patients who have a cytogenetic relapse with frontline TKI therapy. All four TKIs are equivalent if the aim of therapy is to improve survival. In younger patients with high-risk disease and in whom the aim of therapy is to induce a treatment-free remission status, second-generation TKIs may be favored. For CML post-failure on frontline therapy, second-line options include second and third-generation TKIs. Although potent and selective, these TKIs exhibit unique pharmacological profiles and response patterns relative to different patient and disease characteristics, such as patients' comorbidities and financial status, disease stage, and BCR::ABL1 mutational status. Patients who develop the T315I "gatekeeper" mutation display resistance to all currently available TKIs except ponatinib, asciminib, and olverembatinib. Allogeneic stem cell transplantation remains an important therapeutic option for patients with CML-CP and failure (due to resistance) of at least two TKIs and for all patients in advanced-phase disease. Older patients who have a cytogenetic relapse post-failure on all TKIs can maintain long-term survival if they continue a daily most effective/least toxic TKI, with or without the addition of non-TKI anti-CML agents (hydroxyurea, omacetaxine, azacitidine, decitabine, cytarabine, and others).

Evaluation of an In-House Genetic Testing Method for Confirming Prader-Willi and Angelman Syndromes in Sri Lanka.

Prader-Willi syndrome (PWS, MIM 176,270) and Angelman syndrome (AS, MIM 105,830) are caused by imprinting defects of chromosome 15q11-13, with loss of maternal gene expression causing AS and paternal gene expression causing PWS. The diagnosis, once established in most cases by using a methylation-specific PCR test, enables appropriate therapeutic interventions and avoids the need for further investigations. Genetic testing for PWS/AS is limited in Sri Lanka (and in other low- and middle-income countries), mainly because parents are unable to pay for testing as these are not funded by the health service. Ninety cases (46 female) with clinical features suggesting PWS (n = 37) and AS (n = 53), referred by a pediatric endocrinologist and a pediatric neurologist, were recruited. Clinical information and blood samples were obtained following informed consent. DNA was extracted and methylation-specific PCR (MS-PCR) was performed following bisulfite modification of DNA by using an in-house method and a kit. Results were validated using known positive controls. Parent-child trio DNA samples were used in cases with confirmed PWS and AS to determine if the disease was due to a deletion or uniparental disomy. The cost of the MS-PCR testing of the two modification methods and the microsatellite analysis was determined. Among the suspected PWS cases, 19/37 were positive, while 5/53 of the suspected AS cases were positive. The lower identification rate of AS is probably related to the overlap of clinical features of this condition with other disorders. The kit-based modification method was more reliable, less time-consuming, and cost-effective in our laboratory. The kit-based modification followed by MS-PCR described in this study enables more affordable genetic testing of suspected PWS/AS cases, and this is likely to improve patient care by targeting appropriate therapy for the affected cases. Parental genetic counselling is made possible regarding the low recurrence risk, especially where a deletion or uniparental disomy is confirmed. In MS-PCR, negative cases with a strong clinical suspicion of AS, UBE3A mutation testing is required. In addition, imprinting center mutation/deletion testing may also be needed in strongly clinically suspected, MS-PCR negative PWS and AS cases.

Comprehensive genomic analysis reveals clonal origin and subtype-specific evolution in a case of sporadic multiple meningiomas.

Meningioma is the most common primary intracranial tumor in adults, with up to 10% manifesting as multiple tumors. Data on the genomic and molecular changes in sporadic multiple meningiomas are scarce, leading to ongoing debates regarding their evolutionary processes. A comprehensive genetic analysis of a large number of lesions, including precursor lesions, is necessary to explore these two possible origins: clonal and independent. In the present study, we performed whole-exome sequencing and analyzed somatic single-nucleotide variants (SNVs), insertions/deletions (INDELs), and copy number alterations (CNAs) in a patient with sporadic multiple meningiomas. These meningiomas included two mass-forming lesions of different histological subtypes (transitional and chordoid) and two small meningothelial nests. Genetic analysis revealed CNAs on chromosomes 22q and Y as common abnormalities in the two largest tumors. Furthermore, we identified SNV/INDELs unique to each focus, with NF2 mutation prevalent in the transitional meningioma and CREBBP mutation in the chordoid meningioma. Loss of chromosome 22 was detected in two small meningothelial nests. Overall, we elucidated the clonal origin and subtype-specific evolution of multiple meningiomas in this case. CNAs may serve as the initial driving event in meningioma development.

Neurocognitive profiles of 22q11.2 and 16p11.2 deletions and duplications.

Rare recurrent copy number variants (CNVs) at chromosomal loci 22q11.2 and 16p11.2 are genetic disorders with lifespan risk for neuropsychiatric disorders. Microdeletions and duplications are associated with neurocognitive deficits, yet few studies compared these groups using the same measures to address confounding measurement differences. We report a prospective international collaboration applying the same computerized neurocognitive assessment, the Penn Computerized Neurocognitive Battery (CNB), administered in a multi-site study on rare genomic disorders: 22q11.2 deletions (n = 492); 22q11.2 duplications (n = 106); 16p11.2 deletion (n = 117); and 16p11.2 duplications (n = 46). Domains examined include executive functions, episodic memory, complex cognition, social cognition, and psychomotor speed. Accuracy and speed for each domain were included as dependent measures in a mixed-model repeated measures analysis. Locus (22q11.2, 16p11.2) and Copy number (deletion/duplication) were grouping factors and Measure (accuracy, speed) and neurocognitive domain were repeated measures factors, with Sex and Site as covariates. We also examined correlation with IQ. We found a significant Locus × Copy number × Domain × Measure interaction (p = 0.0004). 22q11.2 deletions were associated with greater performance accuracy deficits than 22q11.2 duplications, while 16p11.2 duplications were associated with greater specific deficits than 16p11.2 deletions. Duplications at both loci were associated with reduced speed compared to deletions. Performance profiles differed among the groups with particularly poor memory performance of the 22q11.2 deletion group while the 16p11.2 duplication group had greatest deficits in complex cognition. Average accuracy on the CNB was moderately correlated with Full Scale IQ. Deletions and duplications of 22q11.2 and 16p11.2 have differential effects on accuracy and speed of neurocognition indicating locus specificity of performance profiles. These profile differences can help inform mechanistic substrates to heterogeneity in presentation and outcome, and can only be established in large-scale international consortia using the same neurocognitive assessment. Future studies could aim to link performance profiles to clinical features and brain function.

Multiregion exome sequencing indicates a monoclonal origin of esophageal spindle-cell squamous cell carcinoma.

Esophageal spindle-cell squamous cell carcinoma (ESS) is a rare biphasic neoplasm composed of a carcinomatous component (CaC) and a sarcomatous component (SaC). However, the genomic origin and gene signature of ESS remain unclear. Using whole-exome sequencing of laser-capture microdissection (LCM) tumor samples, we determined that CaC and SaC showed high mutational commonality, with the same top high-frequency mutant genes, mutation signatures, and tumor mutation burden; paired samples shared a median of 25.5% mutation sites. Focal gains were found on chromosomes 3q29, 5p15.33, and 11q13.3. Altered genes were mainly enriched in the RTK-RAS signaling pathway. Phylogenetic trees showed a monoclonal origin of ESS. The most frequently mutated oncogene in the trunk was TP53, followed by NFE2L2, KMT2D, and MUC16. Prognostic associations were found for CDC27, LRP2, APC, and SNAPC4. Our data highlight the monoclonal origin of ESS with TP53 as a potent driver oncogene, suggesting new targeted therapies and immunotherapies as treatment options. © 2024 The Pathological Society of Great Britain and Ireland.

Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q

Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

Revised Diagnosis From Histiocytic Neoplasm to Optic Chiasm Glioblastoma After Genetic Analysis.

A 46-year-old man presented with left eye blurring. Automated visual field testing showed an incongruous right hemianopia, with sparing of the lower temporal quadrant in the right eye. MRI revealed foci of gadolinium enhancement in the optic chiasm and optic tracts. Serologic testing (including myelin oligodendrocyte glycoprotein and neuromyelitis optica antibodies) and cerebrospinal fluid analysis were negative. Whole-body PET/CT scan found no malignancy. Biopsy of the optic chiasm revealed a moderately cellular neoplasm composed of atypical, discohesive cells with enlarged nuclei, prominent eosinophilic nucleoli, and abundant vacuolated cytoplasm. Immunohistochemical stains for CD68 and S100 were positive, whereas those for GFAP, OLIG2, SOX10, and multiple others were negative, supporting a diagnosis of histiocytic neoplasm. Five weeks later, results became available from next-generation sequencing targeting the coding regions of hundreds of malignancy-associated genes and select introns. Alterations associated with histiocytic neoplasms (i.e. BRAF and MAP2K1 mutations) were absent. However, there was a nonsense mutation in the PTEN gene, a hotspot mutation in the TERT gene promotor, and focal amplifications of the CDK4 and MDM2 genes. Additionally, there was chromosome 6q loss, 7 gain, and 10q loss. Based on these findings, the diagnosis was revised to glioblastoma, IDH-wildtype, CNS WHO grade 4. The patient began treatment with temozolomide while continuing radiation therapy. This case illustrates how next-generation sequencing can at times provide more accurate diagnostic information than standard tissue histopathology.

18q Deletion Syndrome Presenting with Late-Onset Combined Immunodeficiency

Patients with chromosome 18q deletion syndrome generally experience hypogammaglobulinemia. Herein, we describe two patients with chromosome 18q deletion syndrome who presented with late-onset combined immune deficiency (LOCID), which has not been previously reported. Patient 1 was a 29-year-old male with 18q deletion syndrome, who was being managed for severe motor and intellectual disabilities at the Yamabiko Medical Welfare Center for 26 years. Although the patient had few infections, he developed Pneumocystis pneumonia at the age of 28. Patient 2, a 48-year-old female with intellectual disability and congenital malformations, was referred to Tokyo Medical and Dental University Hospital with abnormal bilateral lung shadows detected on her chest radiography. Computed tomography showed multiple lymphadenopathies and pneumonia. A lymph node biopsy of the inguinal region revealed granulomatous lymphadenitis, and a chromosomal examination revealed 18q deletion. Array-based genomic hybridization analysis revealed deletion at 18q21.32-q22.3 for patient 1 and at 18q21.33-qter for patient 2. Immune status work-up of the two patients revealed panhypogammaglobulinemia, decreased number of memory B cells and naïve CD4+ and/or CD8+ cells, reduced response on the carboxyfluorescein diacetate succinimidyl ester T-cell division test, and low levels of T-cell receptor recombination excision circles and Ig κ-deleting recombination excision circles. Consequently, both patients were diagnosed with LOCID. Although patients with 18q deletion syndrome generally experience humoral immunodeficiency, the disease can be further complicated by cell-mediated immunodeficiency, causing combined immunodeficiency. Therefore, patients with 18q deletion syndrome should be regularly tested for cellular/humoral immunocompetence.

Abstract PO-035: Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies

Abstract Ba/F3 is a murine pro-B cell line that is dependent on interleukin-3 (IL-3) for its survival and proliferation. It has been widely used for identifying oncogenes and mechanisms of drug-resistance. Using this cell line, we conducted a genome-wide CRISPR knockout screen to identify genes whose loss can confer IL-3 independence, hence may be implied as tumor suppressor genes (TSGs). We identified several genes that map to human chromosome 6q, a frequent deletion hotspot in B-cell malignancies, that may act as TSGs. One of these putative novel TSGs is Slc35a1, which encodes the membrane solute carrier that facilitates the transport of nucleotide sugar (CMP-sialic acid) into the Golgi apparatus for subsequent sialylation. Interestingly, in addition to Slc35a1, pathway analysis of hits from the CRISPR screen identified several genes in the sialic acid metabolism pathway, Nans, Cmas, and St3gal5. We confirmed that their silencing led to decreased protein sialyation and could confer IL-3-independent cell survival and growth in Ba/F3 cells. We continued to characterize Slc35a1 since its homozygous and heterozygous deletions are seen in 7-40% of different types of B-cell malignancies, including approximately 40% of patients with Diffuse Large B-cell Lymphoma. Slc35a1 deletion in murine and human cell-lines conferred cytokine-independent growth. Tail-vein injection of Slc35a1-silenced Ba/F3 cells resulted in allograft tumor growth presenting with splenomegaly and lymphadenopathy. Using Maackia amurensis lectin II (MAL II) to pull down sialylated proteins for mass spectrometry, we identified proteins that were differentially sialylated in Slc35a1 wild-type and knockout cells, among which are the α and β subunits of the integrin receptor, lymphocyte functional antigen 1 (LFA-1). As integrin desialylation can promote its constitutive activation, we are currently studying the impact of LFA-1 sialylation on B-cell function and transformation. We have also developed mouse models with B-cell specific ablation of Nans and Slc35a1 to further evaluate the role of sialylation on B-cell development and tumorigenesis in vivo. Overall, our study demonstrates that sialylation may function as a tumor suppressive mechanism and serve as a therapeutic target in B-cell cancers. Citation Format: Namratha Sheshadri, Rongrong Li, Muhammad Usama Tariq, Jianliang Shen, Jaeyong Jung, Junrong Yan, Kevin Lu, Zhiyuan Shen, Ping Xie, Wei-Xing Zong. Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-035.

EPEN-21. EXPLORATION OF TUMOR SUPPRESSOR LATS1 LOSS IN POSTERIOR FOSSA GROUP A EPENDYMOMA WITH LOSS OF CHROMOSOME 6Q

Abstract INTRODUCTION Chromosome 6q loss (6q-) has recently been identified as an ultra-high-risk factor in posterior fossa group A ependymoma (PFA). This study seeks to pinpoint the biological mechanism underlying the aggressive biology of PFA 6q-. We hypothesize that loss of chromosome 6q gene large tumor suppressor kinase 1 (LATS1) in PFA 6q- results in increased Hippo pathway activity that drives malignant tumor progression. LATS1 is a key negative regulator of the Hippo pathway which may represent a novel therapeutic vulnerability in 6q- PFA. METHODS We are testing this hypothesis using genomic and transcriptomic analysis of PFA patient samples, and by gene knock-in and pharmaceutical interventions of LATS1 and Hippo pathway components in PFA 6q- models. RESULTS Tumor suppressor LATS1 shows reduced expression in PFA 6q- patient samples. Although complete loss of the entire 6q arm is common in PFA 6q-, partial loss of chromosome 6q is also observed, and in all cases this partial loss encompasses the LATS1 locus on chromosome 6q. We used lentiviral transduction to knock-in LATS1 in PFA 6q- cell lines to determine the effect on tumor growth. This demonstrated loss of viability that was specific for 6q- PFA cell lines versus 6q WT control cell lines. DISCUSSION Data point to LATS1 loss being a key driver of the aggressive phenotype of PFA6q-, implicating the Hippo pathway in PFA 6q- and potential for novel therapeutic strategies.

LGG-07. MULTI-OMICS ANALYSIS IDENTIFIES MOLECULAR DRIVERS AND THERAPEUTIC TARGETS IN DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR WITH 1Q GAIN

Abstract BACKGROUND Diffuse leptomeningeal glioneuronal tumors (DLGNT) with 1q gain (1q+) display aggressive clinical behavior. We sought to better describe the clinical characteristics of patients with DLGNT and identify molecular drivers and therapeutic vulnerabilities of DLGNT with 1q+. METHODS DLGNTs were collected from multiple institutions, sent for methylome array and if quantity-sufficient, were subjected to RNA-seq, proteome, and phosphoproteome profiling. Wherever available, clinical data was collected. RESULTS Twenty DLGNTs (13 with 1q+, 7 without 1q+) were collected. Clinical data was obtained on 15 patients (8 with 1q+, 7 without 1q+). The median progression-free survival (PFS) was 17.5 months for those with 1q+ tumors, compared to 51 months for those without (p=0.058). All with 1q+ tumors (n=4) that received CSI radiotherapy progressed (median 12 months), compared to none without (n=3) (p=0.12). There were no responses (complete or partial) in 1q+ tumors that received chemotherapy (n=8), with 2 progressions on therapy. In tumors without 1q+ that received chemotherapy (n=6), 2 responded (1 complete, 1 partial) and 1 progressed on therapy. For 1q+ tumors that received targeted therapy (n=6), there were no responses and 2 progressions on therapy, compared to 1 response and 1 progression for tumors without (n=3). Gene Set Enrichment Analysis of all differentially methylated regions demonstrated enrichment of genes acting in mRNA processing, transcriptional regulation, p53 signaling, TGF-β receptor signaling, and T cell receptor signaling in 1q+ tumors. It identified genes with transcription factor (TF) binding sites for TFs involved in MAPK, PI3K/AKT/mTOR, WNT, and p53 pathways. Consistent with these results, hypomethylated genes at chromosome 1q in 1q+ tumors are positive regulators of MAPK, PI3K/AKT/mTOR, WNT, and p53 signaling. CONCLUSIONS DLGNT with 1q+ were more refractory to current treatment options. We identified putative molecular drivers in 1q+ tumors. Our findings suggest therapy targeting the multiple affected pathways should be considered.

EPEN-24. 5FU SENSITIVITY IS MEDIATED THROUGH TRISOMY UCK2 EXPRESSION IN 1Q+ PFA EPN

Abstract BACKGROUND Nearly all children with gain of chromosome 1q (1q+) PFA ependymoma (EPN) will die, highlighting the urgent need to develop new treatment strategies for these patients. It has recently been shown that 1q+ creates a p53 mutant-like phenotype through trisomy expression of MDM4, a repressor of p53 signaling. This same study found toxic uracil analogs could reverse trisomy MDM4 effect on p53 and this was mediated through another 1q-trisomy gene UCK2. We and other have previously identified 5-fluorouracil (5FU) as an ependymoma specific inhibitor. In this study, we present the mechanism for 5FU sensitivity. METHODS In this study we utilize 1q+ PFA in vitro models to test the efficacy of combination radiation and 5FU in a preclinical setting. We measured p53 signaling through proteomic array and qRT-PCR for p21, a p53 response gene. RESULTS 5FU enhances radiotherapy in 1q+ PFA cell lines. Specifically, 5FU increases p53 activity mediated by the extra copy of UCK2 located on chromosome 1q in 1q+ PFA. Experimental down regulation of UCK2 resulted in decreased 5FU sensitivity in 1q+ PFA cells. Though not statistically significant, we did see prolong survival and decreased tumor burden in 1q+ PFA orthotopic patient-derived xenograft models treated with focal 2Gyx5days radiation and 5FU. CONCLUSION These results are the first to identify a 1q+ specific therapy approach in PFA EPN. Existing phase 1 studies in pediatrics have already established safety and dose of 5FU, providing a rapid translation into phase 2 trials for children both up front and at recurrence for 1q+ PFA.

EPEN-23. TARGETING TRISOMY MDM4 INDUCES CELL DEATH IN 1Q+ PFA EPENDYMOMA THROUGH REACTIVATION OF THE P53 PATHWAY

Abstract BACKGROUND Gain of chromosome 1q (1q+), gives a grave prognosis in pediatric posterior fossa group A ependymoma (PFA). The prevalence of 1q+ in newly diagnosed PFA stands at 25%, escalating to 50% upon recurrence. Conventional therapeutic modalities, such as complete resection and radiation, exhibit minimal efficacy in cases of 1q+ PFA, and the incorporation of additional chemotherapy in clinical trials has failed to yield substantial improvements. In this investigation, we delineate the trisomy-induced overexpression of the MDM4 gene on chromosome 1q as a driver of malignant progression in 1q+ PFA and a viable therapeutic target via idasanutlin, an MDM4 pathway inhibitor. METHODS We are using genetic and pharmaceutical inhibition of the MDM4/MDM2 pathway high-risk PFA 1q+ in vitro and in vivo models to test the efficacy of idasanutlin in a preclinical setting. Treatment effects are measured utilizing growth assays as well as genomic and transcriptomic analysis. RESULTS Our investigation reveals that two 1q+ in vitro models demonstrate a dose dependent sensitivity to idasanutlin compared to normal brain tissue. Upon exposure to idasnutlin, they also have an increase in p21 expression, a critical marker demonstrating reactivation of the p53 pathway, evidenced at both the gene expression and protein levels. Moreover, we found radiation synergistically enhances the efficacy of idasanutlin. Currently, we are evaluating our in vitro results with combination radiation and idasanutlin treatment in our 1q+ PFA preclinical in vivo models. CONCLUSIONS In summary, our study advocates for the exploration of small molecule inhibitors, with a primary focus on the promising idasanutlin compound, as a therapeutic avenue for addressing the challenge posed by ultra-high-risk 1q+ PFA. The compelling preclinical evidence presented herein lays the groundwork for advancing our understanding of the molecular intricacies underlying this malignancy, paving the way for novel and targeted treatment strategies.

EPEN-14. MOLECULAR MARKERS AND PREDICTORS OF OUTCOME FOR PAEDIATRIC INTRACRANIAL EPENDYMOMA IN THE PROSPECTIVE, MULTICENTRE E-HIT2000 TRIAL AND SUBSEQUENT HIT-REGISTRIES: A POOLED ANALYSIS OF 244 PATIENTS

Abstract BACKGROUND Throughout the past decade, molecular tools have revolutionised ependymoma classification, yet their application has been limited in clinical trial cohorts. METHODS We present the molecular analysis of paediatric ependymoma patients from the E-HIT2000 trial (n = 165), or subsequent HIT2000-interim and I-HIT-MED registries (n = 79) based on DNA-methylation profiling with multi-level classification using the Heidelberg Brain Tumor Classifier v12.5 and the analysis of chromosome 1q, 6q, and CDKN2A copy number status. Prior to further analysis, cases without ependymoma group prediction were excluded (n = 16/244). RESULTS 5-year PFS and OS rates varied by group: EPN-PFA (n=146/228) 45±4% and 76±4%; EPN-PFB (n=19/228) 90±7% and 100%; EPN-ZFTA (n=59/228) 63±7% and 87±5%; and EPN-YAP1 (n=4/228) 50±25% and 100%. EPN-PFA was subclassified into nine subtypes. Poor median PFS was observed for PFA1c (n=22, 5-yr-PFS 32±10%/5-yr-OS 77±9%), PFA1d (n=13, 35±14%/72±14%), PFA1e (n=15, 47±13%/59±13%), and PFA2a (n=16, 27±12%/50±14%). Presence of 1q gain was associated with inferior survival (n = 39/146, 29±8%/65±8%). 6q loss impacted PFS, but not OS (n=8, 25±14%/100%). Among 59 EPN-ZFTA cases, 46 clustered with EPN ZFTA-RELA fused, and 12 clustered within recently described subgroups of EPN-ZFTA (EPN-ZFTA alt., Cluster 1: 1/12, Cluster 2: 11/12). Strikingly, 6/11 EPN ZFTA-alt. (Cluster 2) cases showed infratentorial (4/6) or supratentorial midline location (2/6). Homozygous deletion of CDKN2A (CDKN2A-/-; n=8) was only observed in EPN ZFTA-RELA. Presence of EPN ZFTA-alt. (cluster 2) (5-yr-PFS: 36±15%; p<0.001), and CDKN2A-/- (19±16%; p<0.0001) was associated with inferior PFS compared to EPN ZFTA-RELA CDKN2A-wt (81±6%). OS was only impacted for patients with EPN ZFTA-RELA CDKN2A-/- (5-yr-OS: 38±20%, p>0.0001). CONCLUSIONS We present strong evidence supporting the inclusion of molecular criteria into the next generation of clinical trials for children with ependymoma.

EPEN-08. MULTI-OMICS PROFILES REVEAL UNIQUE DEFINING MOLECULAR FEATURES OF POSTERIOR FOSSA EPENDYMOMA SUBTYPE A FROM INFANTS

Abstract BACKGROUND Ependymoma is a heterogeneous brain cancer with multiple subgroups based on locations and molecular profiles. Tumours of the methylation class, posterior fossa group A (PF-EPN-A) represent the most common and aggressive neoplasms and have a poor outcome. PF-EPN-A tumours in infants (under three years of age) are understudied; this project aimed to characterise these infant tumours. METHODS We collected DNA methylation (n=570) and gene expression (n=56) profiles of PF-EPN-A tumours, and single-cell RNA sequencing data of human fetal cerebellum from published studies. RESULTS Analysis of copy number profiles derived from DNA methylation arrays showed that infants with PF-EPN-A have relatively quiescent genomes when compared to children aged four to eighteen years (mean fraction of changes in infants (n=300) = 0.018 and children (n=270) = 0.055; p-value < 2.2 × 10-16). No recurrent focal amplifications or losses were detected, however, copy number loss involving chromosome arms, 12q (12%), 7q (9.7%), 5p (8.3%), and 16p (6%) were relatively common in infants, while 1q gain, 6q loss, and 22q loss were more frequent in children. When we stratified PF-EPN-A based on copy number alterations, infants with altered genomes were associated with poor outcomes compared to infants with stable genomes (fraction of changes = 0), which in turn showed poor overall survival compared to children with stable genomes. Gene set enrichment analysis using data from microarrays showed that infant tumours were specifically enriched with genes involved in the extracellular matrix organization, interferon gamma signaling, neuronal system, autophagy, and signaling by Interleukins, VEGF and PDGF. Integration with single-cell data from the human fetal cerebellum revealed that infants in PF-EPN-A had significantly higher proportions of glial progenitor cells and decreased granule cell progenitor subpopulations. CONCLUSIONS Together, our study identifies distinct genetic and transcriptional programs among infants in PF-EPN-A tumours, representing opportunities to refine future treatment.

PATH-14. A CLINICAL CONUNDRUM - INFANTILE POSTERIOR FOSSA EPENDYMOMA WITH YAP1:MAML FUSION?

Abstract BACKGROUND Ependymomas of infancy (0-18months) have been reported to comprise distinct clinical, pathological and genetic entities. Young age is typically associated with posterior fossa group A (PF-A) tumours and therefore a poor prognosis. Treatment of these infants is challenging with limited therapeutic options that have significant long-term sequelae. METHOD We report a single case of a 4-month old baby girl who presented with obstructive hydrocephalus and a presumed posterior fossa mass. RESULTS Imaging demonstrated a large fairly well-defined solid lesion with multiple T1 high and corresponding T2 intermediate signal intensity foci scattered throughout the lesion and marked mass effect on the cerebellum and fourth ventricle. The lesion was inseparable from the pineal gland, habenula and tectum and only mild diffusion restriction. Whilst initially considered to be a posterior fossa tumour, the presence of a claw sign with the tectal plate, favoured a tectal origin. Histological appearances, together with immunohistochemistry findings (GFAP positive and EMA patchy perinuclear dot expression) were consistent with classic ependymoma. Grading was difficult as mitotic activity was relatively low, although focal areas of high ki67 proliferation index were noted suggesting at least focal grade 3 areas. Retained expression of tumour cells for H3K27me3 on immunohistochemistry suggested the likely posterior fossa group B (PF-B) ependymoma. Local SNP chromosome microarray found no acquired clonal abnormality; with no detection of gain chromosome 1q; loss/LOH 6q; Loss/LOH 9p21.3 [CDKN2A, CDKN2B], chromothripsis 11q [ZFTA::RELA], Loss/LOH 17p13 [TP53] or Loss/LOH 22q [NF2]. Additional NGS sequencing identified a YAP1::MAML2 fusion (confirmed with targeted RT-PCR followed by subsequent Sanger sequencing). CONCLUSION This case highlights the challenges with overlapping clinical, pathological and radiological features in an infant with ependymoma, necessitating a molecular diagnosis. After 21months, the patient remains in complete remission following surgery alone.

A Multimodal Drug-Diet-Immunotherapy Combination Restrains Melanoma Progression and Metastasis.

The genetic landscape of cancer cells can lead to specific metabolic dependencies for tumor growth. Dietary interventions represent an attractive strategy to restrict the availability of key nutrients to tumors. In this study, we identified that growth of a subset of melanoma was severely restricted by a rationally designed combination therapy of a stearoyl-CoA desaturase (SCD) inhibitor with an isocaloric low-oleic acid diet. Despite its importance in oncogenesis, SCD underwent monoallelic codeletion along with PTEN on chromosome 10q in approximately 47.5% of melanoma, and the other SCD allele was methylated, resulting in very low-SCD expression. Although this SCD-deficient subset was refractory to SCD inhibitors, the subset of PTEN wild-type melanoma that retained SCD was sensitive. As dietary oleic acid could potentially blunt the effect of SCD inhibitors, a low oleic acid custom diet was combined with an SCD inhibitor. The combination reduced monounsaturated fatty acids and increased saturated fatty acids, inducing robust apoptosis and growth suppression and inhibiting lung metastasis with minimal toxicity in preclinical mouse models of PTEN wild-type melanoma. When combined with anti-PD1 immunotherapy, the SCD inhibitor improved T-cell functionality and further constrained melanoma growth in mice. Collectively, these results suggest that optimizing SCD inhibitors with diets low in oleic acid may offer a viable and efficacious therapeutic approach for improving melanoma treatment. Significance: Blockade of endogenous production of fatty acids essential for melanoma combined with restriction of dietary intake blocks tumor growth and enhances response to immunotherapy, providing a rational drug-diet treatment regimen for melanoma.