Copy Number Loss Research Articles

BIOM-42. RESPONSE TO DABRAFENIB/TRAMETINIB IN A PATIENT WITH WHO-CNS GRADE 3 BRAF V600E-MUTATED PLEOMORPHIC XANTHOASTROCYTOMA

Abstract BACKGROUND Pleomorphic xanthoastrocytoma (PXA) is a rare central nervous system tumor. Mutations in BRAF V600E are present in 60-80% of cases. While traditionally managed with surgical resection and adjuvant radiation, emerging evidence suggests targeted therapies may be effective, particularly in cases harboring the BRAF V600E mutation. CASE SUMMARY A 35-year-old male with no significant medical history presented in November 2023 with diplopia and headache leading to the discovery of a large left temporal/parietal tumor. A partial resection was performed. Initial histopathological diagnosis was glioblastoma (GBM); thus, the patient was treated with concurrent radiation and temozolomide for 6 weeks. Molecular pathological analysis demonstrated the presence of BRAF V600E mutation and CDKN2A/B copy number loss. Methylation-based tumor profiling was performed at the National Cancer Institute, with composite methylation profile indicating a consensus match to pleomorphic xanthoastrocytoma. Following chemoradiation, a repeat brain MRI in March 2024 showed tumor progression, prompting the initiation of dabrafenib and trametinib therapy. After 1 month of targeted treatment, a repeat brain MRI demonstrated a partial response with a decrease in tumor size and enhancement. In April 2024, he underwent a planned repeat resection. Symptomatically, the patient experienced intermittent headaches and nausea that were responsive to oral steroids with no serious adverse effects to targeted therapy. DISCUSSION This case highlights the therapeutic potential of double BRAF/MEK blockade with dabrafenib and trametinib in BRAF V600E-mutant anaplastic PXA. The observed partial response on imaging suggests the efficacy of targeted therapy in this molecular subtype. These findings underscore the importance of molecular profiling in guiding treatment decisions.

NCOG-50. LONG TERM-SURVIVAL OF AN ELDERLY PATIENT WITH GIANT CELL CEREBELLUM GLIOBLASTOMA (GBM)

Abstract INTRODUCTION Primary cerebellar glioblastoma (GBM) in adult is very rare and accounts for 1% of all GBM. Giant cell variant cerebellar GBM is even rarer. Because of the rarity, its molecular feature, the response to the standard treatment, and biological behavior of tumor progression are not well understood. CASE REPORT An 80 yo male presented with headache, nausea, vomiting and ataxia in 2019. Brain MRI was notable for a large enhancing cerebellar mass. Surgical resection was completed, and pathology was giant cell GBM. Molecular studies showed IDH-wildtype, CDKN2A/B copy number loss, positive TP53 G262V mutation as well as KIT and KDR amplification. The patient underwent standard 6-week concomitant temozolomide (TMZ) and radiation therapy. He only completed 3 cycles of low dose adjuvant temozolomide and two cycles of lomustine due to intolerance of both chemotherapy agents. After that the patient has been off treatment and received brain MRI surveillance over past 4 years. Two years later, his brain MRI showed new punctuate lesions in the radiation field. Suspecting the radiation necrosis; the patient underwent observation. Later, more nodular enhancing lesions developed at the outside region of the radiation field. Tumor progression was considered but the patient wished no biopsy or re-radiation. The new small nodular lesions on MRIs over past two years have behaved unusually with small fluctuations among mild progression, stabilization, or regression. His physical functions have been preserved with a Karnofsky Performance Status scale of 90% with normal cognition. DISCUSSION The patient, who is now 85 years old, has surpassed a 4.5-year survival mark, which is remarkable compared to the supratentorial and the cerebellar GBM with a few months’ survival mark in elderly patients. Our experience emphasizes the different behavior (good response) of a very rare giant cell cerebellar GBM to the standard treatment for newly diagnosed GBM.

Fibroblast Heterogeneity in Hepatocellular Carcinoma and Identification of Prognostic Markers Based on Single-cell Transcriptome Analysis.

HCC is a malignant tumor with high morbidity and mortality. Fibroblasts play a key role in the tumor microenvironment (TME). However, the transcriptional regulatory mechanisms of fibroblasts remained unclear in HCC. The aim of this study was to explore the complex role of fibroblasts in hepatocellular carcinoma (HCC) and to reveal their transcriptional regulatory mechanisms. The goal of this study was to discover potential prognostic markers for HCC by analyzing the genetic variations and differentiation process of fibroblasts. Single-cell transcriptome data from the non-tumor liver site and primary tumor site of HCC were acquired from GSE149614, processed, and clustered using the Seurat pipeline. The inferCNV algorithm was applied to infer copy number variations (CNVs) in fibroblasts. Subsequently, the mechanism underlying the interaction between fibroblasts and other cells in the TME of HCC was analyzed using CellChat software. The trajectory of cellular differentiation of fibroblasts from normal state to malignant state was examined using Monocle 2. SCENIC analysis was performed to identify key transcription factors (TFs) in fibroblasts and assess their correlation with HCC prognosis. Finally, qRT-PCR and Transwell assays were carried out to analyze the mRNA expression and cell metastasis. We identified a total of nine different cell types (B cells, cycling cells, endothelial cells, epithelial cells, fibroblasts, hepatocytes, macrophages, plasma cells, and T cells) based on the single-cell transcriptomic data of HCC. Among them, fibroblasts were highly enriched at the primary tumor site, and their number increased with advanced stages. In addition, significant deletions were detected on chromosome 6p of fibroblasts, and genes in this region were remarkably enriched in pathways associated with antigen processing and presentation. Intercellular communication showed that epithelial cells regulated fibroblasts the most. The differentiation of fibroblasts was mainly accompanied by a transition from normal to malignant state. Importantly, CEBPD and FOSB, the TFs most associated with the putative timing of fibroblasts, were under-expressed in human hepatocytes and showed a significant correlation with HCC prognosis. Overexpressed CEBPD inhibited HCC cell migration and invasion. In conclusion, our study revealed that fibroblast recruitment and differentiation, as well as copy number loss at chromosome 6p, were associated with a higher degree of malignancy and immune dysfunction in HCC. The current discoveries provided new insights into the clinical treatment and diagnosis of HCC.

Malignant Peripheral Nerve Sheath Tumor (MPNST) With Smooth Muscle Differentiation of the Uterus-A Case Report With Emphasis on Diagnostic Pitfalls and Value of DNA Methylation Analysis.

With no more than two dozen cases reported in the literature, malignant peripheral nerve sheath tumor (MPNST) is a rare primary mesenchymal neoplasm arising in the female genital tract. Most cases occurred in middle-aged adults with high grade histology, unfavorable clinical outcome, and no history of neurofibromatosis type 1. Its extreme rarity in this site no doubt poses a diagnostic challenge during routine clinical practice. In the following, we report an additional case of uterine MPNST occurring in a 49-year-old Chinese woman, which was initially misdiagnosed as a leiomyosarcoma. The primary tumor showed two distinctive components-a high-grade poorly differentiated component with markedly pleomorphic spindle cells arranged in a peritheliomatous pattern; and a leiomyosarcoma-like (LMS-like) component with tumor cells displaying obvious myoid differentiation. The patient suffered a recurrence less than 2 years later with the recurrent tumor demonstrating similar features to the high-grade component of the primary tumor. The patient eventually succumbed 46 months later after developing another recurrence despite receiving targeted therapy and chemotherapy. On retrospective molecular analysis, no clinically relevant fusion transcript was detected on RNA sequencing. Interestingly instead, DNA methylation analysis showed the tumor clustered with the "MPNST" group in the German Cancer Research Center (DKFZ) sarcoma classifier. The tumor was also found to have EED gene homozygous deletion, multiple copy number alterations and loss of H3K27me3 expression in both high-grade and LMS-like components. Combining histology with all the ancillary tests results, the diagnosis was most consistent with MPNST. Our case highlights the diagnostic pitfalls for MPNST arising in the female genital tract and the potential clinical utility of DNA methylation analysis.

Metastatic Mesothelioma of the Tunica Vaginalis Presenting as Scrotal and Abdominal Nodules: A Case Report and Review of the Literature.

Mesothelioma of the tunica vaginalis testis (MMTVT) is a rare neoplasm comprising <3% of all cases of malignant mesothelioma (MM). MMTVT derives from the tunica vaginalis testis, an outpouching of the mesothelial-lined abdominal peritoneum that detaches from the abdominal cavity after the descent of the testis. Similar to pleural mesothelioma, asbestos exposure is a known risk factor. However, MMTVT has a better prognosis than pleural mesothelioma. Cutaneous metastases from MMTVT are exceedingly rare. Herein, we describe a case of a 67-year-old man with a history of asbestos exposure presenting with scrotal pain and indurated plaques on his lower abdomen and scrotum. Histologic sections showed a sheet-like dermal proliferation comprising epithelioid cells with necrosis and increased mitotic activity. The clinical and histologic differential diagnosis was broad, including metastatic carcinoma, melanoma, sarcoma, germ cell tumor, hematologic malignancy, neuroendocrine carcinoma, and malignant mesothelioma. By immunohistochemistry, the neoplastic cells were positive for WT1, D2-40, and AE1/AE3, with rare positivity for calretinin, consistent with a diagnosis of mesothelioma. Additional immunohistochemical studies provided no support for the other diagnostic considerations listed above. BAP1 showed retained nuclear expression (normal) by immunohistochemistry. A DNA sequencing panel identified copy number losses in CDKN2A, MTAP, CDKN2B, and NF2, which are frequently identified genetic alterations in malignant mesothelioma. Subsequent testicular imaging demonstrated a diffusely thickened scrotal wall with an enlarged left testicle. Overall, this represents a case of malignant mesothelioma presenting with cutaneous metastases to the scrotum and lower abdomen, with clinical and imaging features suggestive of primary MMTVT. The International Mesothelioma Interest Group recommends using at least 2 mesothelial markers, such as calretinin, WT1, CK5/6 or D2-40, and 2 epithelial markers, such as claudin-4, CEA, MOC-31, as well as a broad-spectrum cytokeratin stain (AE1/AE3) as part of an initial immunohistochemical panel. Metastatic mesothelioma should be included in the differential diagnosis of malignant epithelioid dermal tumors with unusual staining patterns.

Multiomic and clinical analysis of multiply recurrent meningiomas reveals risk factors, underlying biology, and insights into evolution.

An important subset of meningiomas behaves aggressively and is characterized by multiple recurrences. We identify clinical, genetic, and epigenetic predictors of multiply recurrent meningiomas (MRMs) and evaluate the evolution of these meningiomas in patient-matched samples. On multivariable binomial logistic regression, MRMs were significantly associated with male sex (P = 0.012), subtotal resection (P = 0.001), higher number of meningiomas on presentation (P = 0.017), and histopathological sheeting (P = 0.002). Multiomic analysis of primary meningiomas revealed that MRMs have greater copy number losses (P = 0.0313) and increased DNA methylation (P = 0.0155). In meningioma cells with knockdown of EDNRB, a locus with greater promoter methylation and decreased gene expression in MRMs had increased proliferation (P < 0.0001). MRM recurrences were found to be similar to primaries but have a greater burden of copy number gains (P < 0.0001) and increased methylation (P = 0.0045). This clinical and multiomic investigation of MRMs harbors implications for the future development of biomarkers and therapeutic agents for these challenging tumors.

Malignant Peripheral Nerve Sheath Tumor of the Pancreas with Molecular Confirmation: A Case Report with Comprehensive Histopathologic and Molecular Characterization

Abstract Introduction/Objective Malignant peripheral nerve sheath tumors (MPNSTs) are rare soft tissue sarcomas arising from a peripheral nerve or exhibiting nerve sheath differentiation. They are often associated with neurofibromatosis type 1, and most often arise in the trunk, extremities, or head and neck region. In patients without neurofibromatosis type 1, sporadic de novo or radiotherapy-associated MPNSTs can pose a diagnostic challenge. MPNSTs arising from visceral organs are exceedingly rare. Here we report a case of MPNST arising in the pancreas. Methods/Case Report The patient is a 75-year-old female, MUTYH mutation carrier, with prior history of breast and bladder cancers, each treated with respective local excision and radiation therapy. Magnetic resonance imaging (MRI) performed for pancreatic cancer screening showed a 4.7 cm heterogeneous mass in the pancreatic tail suggestive of pancreatic neoplasm. Fine needle aspiration cytology was non-diagnostic. However, due to the radiologic concern for malignancy, the patient proceeded with surgical resection by distal pancreatectomy with splenectomy. Macroscopically, a 6.5 cm, tan-white, partially necrotic mass was identified in the pancreatic tail. Microscopic examination showed a spindle cell neoplasm arranged in a vague fascicular fashion with moderate nuclear pleomorphism, geographic necrosis, and frequent mitotic figures (15 per 10 high power fields). Fifteen lymph nodes were negative for metastasis. The tumor was confined to the pancreas grossly and microscopically. Immunohistochemical stains showed the neoplastic spindle cells to be positive for TLE1 (diffuse) and S100 (patchy), while negative for SOX10, CD117, DOG1, desmin, actin, MyoD1, ERG, STAT6, and ALK1. In addition, H3K27me3 showed loss of nuclear expression. Next generation sequencing (NGS) revealed gene mutations in NF1, TP53, PIK3CA, and PTEN, and copy number losses involving CDKN2A and CDKN2B. Genome-wide DNA methylation analysis was performed which revealed copy number losses of CDKN2A/B and gains of PTCH1 and MDM2. However, the Sarcoma DNA Methylation Classifier did not provide any diagnostic matches. Overall, the morphologic features, loss of H3K27me3 expression, presence of NF1 and TP53 mutations, and copy number losses in CDKN2A/B supported a diagnosis of high-grade MPNST. The patient has been followed clinically without adjuvant therapy. Results (if a Case Study enter NA) N/A Conclusion We report an extremely rare case of MPNST of the pancreas with molecular confirmation.

Comprehensive mutational profiling identifies new driver events in cutaneous leiomyosarcoma.

Cutaneous leiomyosarcoma (cLMS) is a rare soft tissue neoplasm, showing smooth muscle differentiation, that arises from the mesenchymal cells of the dermis. To-date, genetic investigation of these tumours has involved studies with small sample sizes and limited analyses that identified recurrent somatic mutations in RB1 and TP53, copy number gain of MYCOD and IGF1R, and copy number loss of PTEN. To better understand the molecular pathogenesis of cLMS, we comprehensively explored the mutational landscape of these rare tumours to identify candidate driver events. In this retrospective, multi-institutional study, we performed whole-exome sequencing and RNA sequencing on 38 cases of cLMS. TP53 and RB1 were identified as significantly mutated, thus, represent validated driver genes of cLMS. COSMIC mutational signatures SBS7a/b and DBS1 were recurrent, thus, ultraviolet light exposure may be an aetiological factor driving cLMS. Analysis of significantly recurrent somatic copy number alterations, which represent candidate driver events, found focal (<10Mb) deletions encompassing TP53 and KDM6B, and amplifications encompassing ZMYM2, MYOCD, MAP2K4 and NCOR1. A larger (24 Mb) recurrent deletion encompassing CYLD was also identified as significant. Significantly recurrent broad copy number alterations, involving at least half of a chromosome arm, included deletions of 6p/q, 10p/q, 11q, 12q, 13q and 16p/q, and amplification of 15q. Notably PTEN is located on 10q, RB1 on 13q and IGFR1 on 15q. Fusion gene analysis identified recurrent CRTC1/3::MAML2 fusions, as well as many novel fusions in individual samples. Our analysis of the largest number of cLMS cases to-date highlights the importance of large cohort sizes and the exploration beyond small targeted gene panels when performing molecular analyses, as it allowed a comprehensive exploration of the mutational landscape of these tumours and identification of novel candidate driver events. It also uniquely afforded the opportunity to compare the molecular phenotype of cLMS with LMS of other tissue types, such as uterine and soft tissue LMS. Given that molecular profiling has resulted in the development of novel targeted treatment approaches for uterine and soft tissue LMS, our study now allows the same opportunities to become available for patients with cLMS.

12312 A Comparative Genomic Analysis Between Parathyroid Adenomas And Carcinomas With Heterozygous Germline Cdc73 Mutations

Abstract Disclosure: Y. Li: None. W.F. Simonds: None. H. Chen: None. Introduction: Parathyroid cancer is responsible for ∼1% of primary hyperparathyroidism. The hereditary form of this malignancy has been linked to germline mutations of the CDC73 gene. We previously reported that only the germline CDC73 mutations causing a significant impact on the function/expression of its encoded protein are associated with parathyroid carcinomas. On the other hand, these germline mutations may lead to only parathyroid adenomas or no parathyroid disease, suggesting incomplete penetrance. This observation raises the question of what additional genomic events are required for parathyroid tumorigenesis in carriers with germline CDC73 mutations. Methods: Twelve patients harboring heterozygous germline CDC73 mutations were included in this study. Whole exome sequencing was performed in all 12 parathyroid tumors (6 adenomas and 6 carcinomas) and the paired normal tissues. RNA sequencing was successfully performed in three of each parathyroid carcinomas, adenomas, and normal glands. The sequencing raw data were processed using the CCBR-Pipeliner. EnrichR, Gene Set Enrichment Analysis, and CIBERSORT were used for the downstream analyses. Results: All parathyroid carcinomas and adenomas had gained one somatic event predicted to cause a complete inactivation of the second CDC73 allele. Several distinctive genomic features were identified in parathyroid carcinomas compared to adenomas, including more single nucleotide variants bearing the signature of APOBEC deamination, frequent mutations among the genes involved in the PI-3K/mTOR signaling, and substantially more copy number variations and differentially expressed genes. Parathyroid carcinomas also share some genomic features with adenomas. For instance, both tumors have frequent somatic mutations and copy number loss that impact the genes related to T-cell receptor signaling and tumor antigen presentation, suggesting a shared strategy to evade immune surveillance. Consistent with these findings, an in silico estimation of immune cell composition in tumors found an absence of CD8+ T-cells in parathyroid adenomas and carcinomas. Conclusion: Our study provides evidence that biallelic inactivation of CDC73 is essential for parathyroid tumorigenesis in carriers with germline mutations of this gene. Despite sharing some genomic features with adenomas, parathyroid carcinomas have more distinctive alterations in the genome, some of which may be critical for cancer formation. Presentation: 6/2/2024

12312 A Comparative Genomic Analysis Between Parathyroid Adenomas And Carcinomas With Heterozygous Germline Cdc73 Mutations

Abstract Disclosure: Y. Li: None. W.F. Simonds: None. H. Chen: None. Introduction: Parathyroid cancer is responsible for ∼1% of primary hyperparathyroidism. The hereditary form of this malignancy has been linked to germline mutations of the CDC73 gene. We previously reported that only the germline CDC73 mutations causing a significant impact on the function/expression of its encoded protein are associated with parathyroid carcinomas. On the other hand, these germline mutations may lead to only parathyroid adenomas or no parathyroid disease, suggesting incomplete penetrance. This observation raises the question of what additional genomic events are required for parathyroid tumorigenesis in carriers with germline CDC73 mutations. Methods: Twelve patients harboring heterozygous germline CDC73 mutations were included in this study. Whole exome sequencing was performed in all 12 parathyroid tumors (6 adenomas and 6 carcinomas) and the paired normal tissues. RNA sequencing was successfully performed in three of each parathyroid carcinomas, adenomas, and normal glands. The sequencing raw data were processed using the CCBR-Pipeliner. EnrichR, Gene Set Enrichment Analysis, and CIBERSORT were used for the downstream analyses. Results: All parathyroid carcinomas and adenomas had gained one somatic event predicted to cause a complete inactivation of the second CDC73 allele. Several distinctive genomic features were identified in parathyroid carcinomas compared to adenomas, including more single nucleotide variants bearing the signature of APOBEC deamination, frequent mutations among the genes involved in the PI-3K/mTOR signaling, and substantially more copy number variations and differentially expressed genes. Parathyroid carcinomas also share some genomic features with adenomas. For instance, both tumors have frequent somatic mutations and copy number loss that impact the genes related to T-cell receptor signaling and tumor antigen presentation, suggesting a shared strategy to evade immune surveillance. Consistent with these findings, an in silico estimation of immune cell composition in tumors found an absence of CD8+ T-cells in parathyroid adenomas and carcinomas. Conclusion: Our study provides evidence that biallelic inactivation of CDC73 is essential for parathyroid tumorigenesis in carriers with germline mutations of this gene. Despite sharing some genomic features with adenomas, parathyroid carcinomas have more distinctive alterations in the genome, some of which may be critical for cancer formation. Presentation: 6/2/2024

Targeted dynamic phospho-proteogenomic analysis of gastric cancer cells suggests host immunity provides survival benefit

Despite of massive emergence of molecular targeting drugs, the mainstay of advanced gastric cancer (GC) therapy is DNA-damaging drugs. Using a reverse-phase protein array-based proteogenomic analysis of a panel of eight GC cell lines, we identified genetic alterations and signaling pathways, potentially associated with resistance to DNA-damaging drugs, including 5-fluorouracil (5FU), cisplatin, and etoposide. Resistance to cisplatin and etoposide, but not 5FU, was negatively associated with global copy number loss, vimentin expression, and caspase activity, which are considered hallmarks of previously established EMT subtype. The segregation of 19,392 protein expression time courses by sensitive and resistant cell lines for the drugs tested revealed that 5FU-resistant cell lines had lower changes in global protein dynamics, suggesting their robust protein level regulation, compared to their sensitive counterparts, whereas the cell lines that are resistant to other drugs showed increased protein dynamics in response to each drug. Despite faint global protein dynamics, 5FU-resistant cell lines showed increased STAT1 phosphorylation and PD-L1 expression in response to 5FU. In publicly available cohort data, expression of STAT1 and NFκB target genes induced by proinflammatory cytokines was associated with prolonged survival in GC. In our validation cohort, total lymphocyte count (TLC), rather than PD-L1 positivity, predicted a better relapse-free survival rate in GC patients with 5FU-based adjuvant chemotherapy than those with surgery alone. Moreover, TLC+ patients who had no survival benefit from adjuvant chemotherapy were discriminated by expression of IκBα, a potent negative regulator of NFκB. Collectively, our results suggest that 5FU resistance observed in cell lines may be overcome by host immunity or by combination therapy with immune checkpoint blockade.

Focal deletions of a promoter tether activate the IRX3 oncogene in T-cell acute lymphoblastic leukemia

AbstractOncogenes can be activated in cis through multiple mechanisms including enhancer hijacking events and noncoding mutations that create enhancers or promoters de novo. These paradigms have helped parse somatic variation of noncoding cancer genomes, thereby providing a rationale to identify noncanonical mechanisms of gene activation. Here we describe a novel mechanism of oncogene activation whereby focal copy number loss of an intronic element within the FTO gene leads to aberrant expression of IRX3, an oncogene in T-cell acute lymphoblastic leukemia. Loss of this CTCF-bound element downstream to IRX3 (+224 kb) leads to enhancer hijack of an upstream developmentally active super-enhancer of the CRNDE long noncoding RNA (−644 kb). Unexpectedly, the CRNDE super-enhancer interacts with the IRX3 promoter with no transcriptional output until it is untethered from the FTO intronic site. We propose that “promoter tethering” of oncogenes to inert regions of the genome is a previously unappreciated biological mechanism preventing tumorigenesis.

Multiple Myeloma Risk and Outcomes Are Associated with Pathogenic Germline Variants in DNA Repair Genes.

First-degree relatives of patients with multiple myeloma are at increased risk for the disease, but the contribution of pathogenic germline variants (PGV) in hereditary cancer genes to multiple myeloma risk and outcomes is not well characterized. To address this, we analyzed germline exomes in two independent cohorts of 895 and 786 patients with multiple myeloma. PGVs were identified in 8.6% of the Discovery cohort and 11.5% of the Replication cohort, with a notable presence of high- or moderate-penetrance PGVs (associated with autosomal dominant cancer predisposition) in DNA repair genes (3.6% and 4.1%, respectively). PGVs in BRCA1 (OR = 3.9, FDR < 0.01) and BRCA2 (OR = 7.0, FDR < 0.001) were significantly enriched in patients with multiple myeloma when compared with 134,187 healthy controls. Five of the eight BRCA2 PGV carriers exhibited tumor-specific copy number loss in BRCA2, suggesting somatic loss of heterozygosity. PGVs associated with autosomal dominant cancer predisposition were associated with younger age at diagnosis, personal or familial cancer history, and longer progression-free survival after upfront high-dose melphalan and autologous stem-cell transplantation (P < 0.01). Significance: Our findings suggest up to 10% of patients with multiple myeloma may have an unsuspected cancer predisposition syndrome. Given familial implications and favorable outcomes with high-dose melphalan and autologous stem-cell transplantation in high-penetrance PGV carriers, genetic testing should be considered for young or newly diagnosed patients with a personal or family cancer history. See related commentary by Walker, p. 375.

Midnolin, a Genetic Risk Factor for Parkinson's Disease, Promotes Neurite Outgrowth Accompanied by Early Growth Response 1 Activation in PC12 Cells.

Parkinson's disease (PD) is an age-related progressive neurodegenerative disease. Previously, we identified midnolin (MIDN) as a genetic risk factor for PD. Although MIDN copy number loss increases the risk of PD, the molecular function of MIDN remains unclear. To investigate the role of MIDN in PD, we established monoclonal Midn knockout (KO) PC12 cell models. Midn KO inhibited neurite outgrowth and neurofilament light chain (Nefl) gene expression. Although MIDN is mainly localized in the nucleus, it does not encode DNA-binding domains. We therefore hypothesized that MIDN might bind to certain transcription factors and regulate gene expression. Of the candidate transcription factors, we focused on early growth response 1 (EGR1) because it is required for neurite outgrowth and its target genes are downregulated by Midn KO. An interaction between MIDN and EGR1 was confirmed by immunoprecipitation. Surprisingly, although EGR1 protein levels were significantly increased in Midn KO cells, the binding of EGR1 to the Nefl promoter and resulting transcriptional activity were downregulated as measured by luciferase assay and chromatin immunoprecipitation quantitative real-time polymerase chain reaction. Overall, we identified the MIDN-dependent regulation of EGR1 function. This mechanism may be an underlying reason for the neurite outgrowth defects of Midn KO PC12 cells.

Clinical and methylomic features of spinal meningiomas.

The objective of our study was to analyze methylomic and clinical features of a cohort of spinal meningiomas (SMs) resected at our institution. This is a retrospective study of patients undergoing SM resection at our institution between 2010 and 2023. Clinical and radiographic characteristics were reviewed and analyzed with standard statistical methods. A Partitioning Around Medoids approach was used to cluster SMs with methylation data in a combined cohort from our institution and a publicly available dataset by methylation profiles. Clinical variables and pathway analyses were compared for the resulting clusters. Sixty-five SMs were resected in 53 patients with median radiographic follow-up of 34 months. Forty-six (87%) patients were female. The median age at surgery was 65 years and median tumor diameter was 1.9cm. The five-year progression-free survival rate was 90%, with subtotal resection being associated with recurrence or progression (p = .017). SMs clustered into hypermethylation, intermediate methylation, and hypomethylation subgroups. Tumors in the hypermethylated subgroup were associated with higher WHO grade (p = .046) and higher risk histological subtypes (p <.001), while tumors in the hypomethylated subgroup were least likely to present with copy-number loss in chromosome 22q (p <.0001). SMs classified as immune-enriched under a previously developed intracranial meningioma classifier did not have increased leukocyte fractions or hypomethylation of genes typically hypomethylated in immune-enriched tumors. SMs are more benign than their intracranial counterparts, and gross-total resection results in long term PFS. Methylation profiling identifies subgroups with differences in clinical variables.

Single-cell detection of copy number changes reveals dynamic mechanisms of adaptation to antifungals in Candida albicans.

Genomic copy number changes are associated with antifungal drug resistance and virulence across diverse fungal pathogens, but the rate and dynamics of these genomic changes in the presence of antifungal drugs are unknown. Here we optimized a dual-fluorescent reporter system in the diploid pathogen Candida albicans to quantify haplotype-specific copy number variation (CNV) and loss of heterozygosity (LOH) at the single-cell level with flow cytometry. We followed the frequency and dynamics of CNV and LOH at two distinct genomic locations in the presence and absence of antifungal drugs in vitro and in a murine model of candidiasis. Copy number changes were rapid and dynamic during adaptation to fluconazole and frequently involved competing subpopulations with distinct genotypes. This study provides quantitative evidence for the rapid speed at which diverse genotypes arise and undergo dynamic population-level fluctuations during adaptation to antifungal drugs in vitro and in vivo.

Loss of copy numbers of retrotransposons (HERVK) on chromosome 7p11.2 impacts EGFR (Epidermal Growth Factor Receptor)-induced phenotypes for platinum sensitivity and long-term survival in ovarian cancer-A study from the OVCAD consortium.

We analyzed variations in the epidermal growth factor receptor (EGFR) gene and 5'-upstream region to identify potential molecular predictors of treatment response in primary epithelial ovarian cancer. Tumor tissues collected during debulking surgery from the prospective multicenter OVCAD study were investigated. Copy number variations in the human endogenous retrovirus sequence human endogenous retrovirus K9 (HERVK9) and EGFR Exons 7 and 9, as well as repeat length and loss of heterozygosity of polymorphic CA-SSR I and relative EGFR mRNA expression were determined quantitatively. At least one EGFR variation was observed in 94% of the patients. Among the 30 combinations of variations discovered, enhanced platinum sensitivity (n = 151) was found dominantly with HERVK9 haploidy and Exon 7 tetraploidy, overrepresented among patients with survival ≥120 months (24/29, p = .0212). EGFR overexpression (≥80 percentile) was significantly less likely in the responders (17% vs. 32%, p = .044). Multivariate Cox regression analysis, including age, FIGO stage, and grade, indicated that the patients' subgroup was prognostically significant for CA-SSR I repeat length <18 CA for both alleles (HR 0.276, 95% confidence interval 0.109-0.655, p = .001). Although EGFR variations occur in ovarian cancer, the mRNA levels remain low compared to other EGFR-mutated cancers. Notably, the inherited length of the CA-SSR I repeat, HERVK9 haploidy, and Exon 7 tetraploidy conferred three times higher odds ratio to survive for more than 10 years under therapy. This may add value in guiding therapies if determined during follow-up in circulating tumor cells or circulating tumor DNA and offers HERVK9 as a potential therapeutic target.

The History of Chromosomal Instability in Genome-Doubled Tumors.

Tumors frequently display high chromosomal instability and contain multiple copies of genomic regions. Here, we describe Gain Route Identification and Timing In Cancer (GRITIC), a generic method for timing genomic gains leading to complex copy number states, using single-sample bulk whole-genome sequencing data. By applying GRITIC to 6,091 tumors, we found that non-parsimonious evolution is frequent in the formation of complex copy number states in genome-doubled tumors. We measured chromosomal instability before and after genome duplication in human tumors and found that late genome doubling was followed by an increase in the rate of copy number gain. Copy number gains often accumulate as punctuated bursts, commonly after genome doubling. We infer that genome duplications typically affect the landscape of copy number losses, while only minimally impacting copy number gains. In summary, GRITIC is a novel copy number gain timing framework that permits the analysis of copy number evolution in chromosomally unstable tumors. Significance: Complex genomic gains are associated with whole-genome duplications, which are frequent across tumors, span a large fraction of their genomes, and are linked to poorer outcomes. GRITIC infers when these gains occur during tumor development, which will help to identify the genetic events that drive tumor evolution. See related commentary by Taylor, p. 1766.

Non-linear transcriptional responses to gradual modulation of transcription factor dosage.

Genomic loci associated with common traits and diseases are typically non-coding and likely impact gene expression, sometimes coinciding with rare loss-of-function variants in the target gene. However, our understanding of how gradual changes in gene dosage affect molecular, cellular, and organismal traits is currently limited. To address this gap, we induced gradual changes in gene expression of four genes using CRISPR activation and inactivation. Downstream transcriptional consequences of dosage modulation of three master trans-regulators associated with blood cell traits (GFI1B, NFE2, and MYB) were examined using targeted single-cell multimodal sequencing. We showed that guide tiling around the TSS is the most effective way to modulate cis gene expression across a wide range of fold-changes, with further effects from chromatin accessibility and histone marks that differ between the inhibition and activation systems. Our single-cell data allowed us to precisely detect subtle to large gene expression changes in dozens of trans genes, revealing that many responses to dosage changes of these three TFs are non-linear, including non-monotonic behaviours, even when constraining the fold-changes of the master regulators to a copy number gain or loss. We found that the dosage properties are linked to gene constraint and that some of these non-linear responses are enriched for disease and GWAS genes. Overall, our study provides a straightforward and scalable method to precisely modulate gene expression and gain insights into its downstream consequences at high resolution.

12 Concordance Analysis of Tissue and Circulating Tumor DNA (ctDNA) in Renal Cell Carcinoma (RCC): Insights from a Multimodal Real-World Database

Abstract Background Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) has emerged as a powerful complement to tissue NGS, offering a noninvasive and serially conductible test. Its application holds promise in enhancing the assessment of spatial and temporal molecular tumor heterogeneity, thus providing valuable insights into cancer progression and treatment response. In this study, we explore the mutational landscape of renal cell carcinoma (RCC) patients through comprehensive profiling of mutations in ctDNA and matched tissue samples, aiming to elucidate the concordance and clinical significance of molecular alterations detected in both circulating and tissue-derived DNA. Methods From the Tempus multimodal database, we retrospectively analyzed de-identified NGS data from patients with RCC that had dual tissue (Tempus xT, 648 genes) and ctDNA testing (Tempus xF, 105 genes). Patients with matched samples (collected +/- 90 days of one another) were included. We evaluated socio-demographic and clinical characteristics and select pathogenic somatic short variants (PSSV) and copy number variants [(amplifications and deletions, two copy number losses (CNL)]. Concordance analyses were restricted to the 105 genes tested on the ctDNA panel and further restricted to short variants, with the exception of amplifications and CNL detected by both xF and xT. Analysis was further stratified by metastatic status (n=260, metastatic, n=120 non-metastatic) prior to collection of both xT and xF. Results Among all patients (n=393), the median age was 61 years, and 71% were male. The patient cohort comprised a diverse population based on race, with 75% white, 12% African American, 4.8% Asian and 8% others. The median time from tissue to blood collection was 21 days (IQR, 7, 39). 67% (n=265) and 68% (n=266) had metastatic disease at the time of tissue and blood collection, respectively. The most common tissue sites were kidney (49%, n=189), bone (11%, n=43), lung (9%, n=34), lymph node (8%, n=29), liver (6%, n=23), and brain/CNS (4%, n=17). Genes harboring the most common PSSV in tissue included VHL (59% n=232), PBRM1 (31%, n=123), SETD2 (23%, n=91), TP53 (14%, n=54), BAP1 (12%, n=46) and TERT (11%, n=45). Genes with common PSSV in ctDNA included TP53 (23%, n=91), VHL (18%, n=69), BAP1 (6%, n=23), PBRM1 (5%, n=21), PTEN (4%, n=15), KRAS (4%, n=14) and NF2 (4%, n=14). The combination of tissue and ctDNA testing increased the detection of mutations (Table 1). There was higher concordance between somatic alterations in select genes among patients with metastases vs. non-metastases, including BAP1 (55.9% vs. 9.1%), TP53 (36.8% vs. 9.1%), VHL (32.3% vs. 12.5%), ARID1A (25% vs. 16.7%), and ATM (25% vs. 0%). Table 1: Detection of genetic mutations in tissue and ctDNA testing Conclusions The analysis conducted in this study highlights the complementary nature of ctDNA profiling alongside tissue-based NGS in RCC, demonstrating an increased detection of mutations. Particularly, we observed a higher concordance between ctDNA and tissue profiling in individuals with metastatic disease, suggesting the potential utility of ctDNA analysis in advanced stages of RCC. Further research is warranted to elucidate how longitudinal ctDNA analysis can delineate biomarkers of response and resistance at both the mutation and ctDNA fraction levels. Understanding these dynamics could offer valuable insights into disease progression and guide personalized treatment strategies for RCC patients. DOD CDMRP Funding: no