Sporadic Patients Research Articles

#2675 Role of ANXA4 as a modifier in VHL-dependent ccRRC

Abstract Background and Aims The von Hippel-Lindau (VHL) syndrome is a rare genetic disease caused by inactivating mutations of the VHL tumor suppressor gene. VHL syndrome patients frequently develop clear cell renal cell carcinomas (ccRCCs). pVHL acts as the substrate recognition subunit of an E3 ligase involved in the ubiquitinylation of HIFs. Consequently, aberrant HIF-signalling results in a pseudo-hypoxic state characterized by pronounced vascularization in respective tumours. Previous studies have indicated VHL-dependent alterations of the extracellular matrix (ECM). However, to date comprehensive description and understanding of VHL-dependent matrisome signatures is missing. Method Proteomics were used to reveal VHL-dependent protein expression in ccRCC cell lines (A498, 786-O) and CRISPR/Cas9-modified human proximal tubular epithelial cells (hRPTECs) as well as syndromic and sporadic ccRCC patient cohorts. For candidate proteins analysis of proteomic and histological datasets was performed. Further functional analysis was conducted using overexpression, CRISPR/Cas9-mediated knockout (KO) and shRNA-mediated knockdown (KD) approaches (assessment of proliferation, migration, invasion, ECM, cellular membrane-integrity, protein localisation studies and transcriptomics). Results Filtering of proteomic datasets (ccRCC tumors and cell lines) for matrisomal proteins highlighted an enrichment of the ANXA-protein family. Interestingly, functional genetic titration of ANXA4 employing knockdown, overexpression or complete ablation using CRISPR/Cas9 genome editing did not translate into major changes of cellular proliferation or migration in renal cancer or tubular cells. Detailed localization studies revealed that ANXA4 is detectable in distinct cellular compartments, namely the cell membrane and nucleus/nuclear lamina. Various cellular stressors (including cellular densities) initiated shuttling of ANXA4 between these different compartments. Remarkably, similar distribution patterns were detectable in situ in tumours tissue of ccRCC patients. Further transcriptomic analysis of ANXA4 knockout cells demonstrated a major impact on ECM- and cell-adhesion-associated gene sets, reflected by impaired invasive migratory capacity in conditions of reduced ANXA4-expression. Conclusion Loss of VHL distinctly alters the ECM composition in ccRCC. We have identified the ECM-associated ANXA4 as a prominently upregulated protein, although canonical hallmarks of cancer were not directly influenced. Based on RNA-sequencing studies and subcellular localization patterns we assume that ANXA4 (co-)regulates matrisome-related transcriptomic signatures in response to various external stressors. These observations highlight the intricate regulation of ECM-associated signalling pathways in the context of VHL-dependent renal cell carcinoma.

Identifying potential (re)hemorrhage among sporadic cerebral cavernous malformations using machine learning

The (re)hemorrhage in patients with sporadic cerebral cavernous malformations (CCM) was the primary aim for CCM management. However, accurately identifying the potential (re)hemorrhage among sporadic CCM patients in advance remains a challenge. This study aims to develop machine learning models to detect potential (re)hemorrhage in sporadic CCM patients. This study was based on a dataset of 731 sporadic CCM patients in open data platform Dryad. Sporadic CCM patients were followed up 5 years from January 2003 to December 2018. Support vector machine (SVM), stacked generalization, and extreme gradient boosting (XGBoost) were used to construct models. The performance of models was evaluated by area under receiver operating characteristic curves (AUROC), area under the precision-recall curve (PR-AUC) and other metrics. A total of 517 patients with sporadic CCM were included (330 female [63.8%], mean [SD] age at diagnosis, 42.1 [15.5] years). 76 (re)hemorrhage (14.7%) occurred during follow-up. Among 3 machine learning models, XGBoost model yielded the highest mean (SD) AUROC (0.87 [0.06]) in cross-validation. The top 4 features of XGBoost model were ranked with SHAP (SHapley Additive exPlanations). All-Elements XGBoost model achieved an AUROCs of 0.84 and PR-AUC of 0.49 in testing set, with a sensitivity of 0.86 and a specificity of 0.76. Importantly, 4-Elements XGBoost model developed using top 4 features got a AUROCs of 0.83 and PR-AUC of 0.40, a sensitivity of 0.79, and a specificity of 0.72 in testing set. Two machine learning-based models achieved accurate performance in identifying potential (re)hemorrhages within 5 years in sporadic CCM patients. These models may provide insights for clinical decision-making.

Monocytes release cystatin F dimer to associate with Aβ and aggravate amyloid pathology and cognitive deficits in Alzheimer’s disease

BackgroundUnderstanding the molecular mechanisms of Alzheimer’s disease (AD) has important clinical implications for guiding therapy. Impaired amyloid beta (Aβ) clearance is critical in the pathogenesis of sporadic AD, and blood monocytes play an important role in Aβ clearance in the periphery. However, the mechanism underlying the defective phagocytosis of Aβ by monocytes in AD remains unclear.MethodsInitially, we collected whole blood samples from sporadic AD patients and isolated the monocytes for RNA sequencing analysis. By establishing APP/PS1 transgenic model mice with monocyte-specific cystatin F overexpression, we assessed the influence of monocyte-derived cystatin F on AD development. We further used a nondenaturing gel to identify the structure of the secreted cystatin F in plasma. Flow cytometry, enzyme-linked immunosorbent assays and laser scanning confocal microscopy were used to analyse the internalization of Aβ by monocytes. Pull down assays, bimolecular fluorescence complementation assays and total internal reflection fluorescence microscopy were used to determine the interactions and potential interactional amino acids between the cystatin F protein and Aβ. Finally, the cystatin F protein was purified and injected via the tail vein into 5XFAD mice to assess AD pathology.ResultsOur results demonstrated that the expression of the cystatin F protein was specifically increased in the monocytes of AD patients. Monocyte-derived cystatin F increased Aβ deposition and exacerbated cognitive deficits in APP/PS1 mice. Furthermore, secreted cystatin F in the plasma of AD patients has a dimeric structure that is closely related to clinical signs of AD. Moreover, we noted that the cystatin F dimer blocks the phagocytosis of Aβ by monocytes. Mechanistically, the cystatin F dimer physically interacts with Aβ to inhibit its recognition and internalization by monocytes through certain amino acid interactions between the cystatin F dimer and Aβ. We found that high levels of the cystatin F dimer protein in blood contributed to amyloid pathology and cognitive deficits as a risk factor in 5XFAD mice.ConclusionsOur findings highlight that the cystatin F dimer plays a crucial role in regulating Aβ metabolism via its peripheral clearance pathway, providing us with a potential biomarker for diagnosis and potential target for therapeutic intervention.

At Early Rheumatoid Arthritis Stage, the Infectious Spectrum Is Driven by Non-Familial Factors and Anti-CCP Immunization.

Background/Objectives: Patients with rheumatoid arthritis (RA) are prone to develop infections. Methods: Accordingly, 195 untreated early (e)RA patients and 398 healthy controls were selected from women in Tatarstan's cohort to study infectious history in the anamnesis (four criteria) and in the previous year (16 criteria). Information about annual infections was collected face-to-face from year to year by a qualified rheumatologist/general practitioner and included the active use of information from medical records. Results: In the anamnesis, tuberculosis, and pneumonia, and in the previous year, respiratory tract infections, skin infections, and herpes simplex virus reactivation incidence were reported to be increased in eRA patients, as well as the event number and duration of acute and chronic tonsillitis. Moreover, more bacterial-suspected upper respiratory infections and urinary tract infections were retrieved in sporadic eRA patients as compared to familial eRA patients. An elevated immunization against CCP prevented respiratory tract infection in those with HSV exacerbation. Finally, associations were retrieved between infection (event number/delay) and RA indices: (i) chronic tonsillitis exacerbations with disease activity and health assessment (HAQ) in familial eRA; (ii) bacterial-suspected upper respiratory infections with the number of swollen and tender joints in sporadic eRA; and (iii) HSV exacerbation with inflammation in eRA patients with negative/low response against CCP. Here, we demonstrate the complex nature of the interplay of RA with specific infections. Conclusions: For the first time, differences in the patterns of annual trivial infections and their links with RA indices were found in cohorts of familial and sporadic cases of the disease. Additionally, for the first time, we identified a remarkable relationship between early RA and exacerbations of chronic tonsillitis, as well as tuberculosis in the patient's history. Altogether, this study supports the existence of a complex interplay between infections and RA at onset driven by familial status and the presence of anti-CCP Ab at elevated levels.

Inhibition of NF-κB with an Analog of Withaferin-A Restores TDP-43 Homeostasis and Proteome Profiles in a Model of Sporadic ALS.

The current knowledge on pathogenic mechanisms in amyotrophic lateral sclerosis (ALS) has widely been derived from studies with cell and animal models bearing ALS-linked genetic mutations. However, it remains unclear to what extent these disease models are of relevance to sporadic ALS. Few years ago, we reported that the cerebrospinal fluid (CSF) from sporadic ALS patients contains toxic factors for disease transmission in mice via chronic intracerebroventricular (i.c.v.) infusion. Thus a 14-day i.c.v. infusion of pooled CSF samples from ALS cases in mice provoked motor impairment as well as ALS-like pathological features. This offers a unique paradigm to test therapeutics in the context of sporadic ALS disease. Here, we tested a new Withaferin-A analog (IMS-088) inhibitor of NF-κB that was found recently to mitigate disease phenotypes in mouse models of familial disease expressing TDP-43 mutant. Our results show that oral intake of IMS-088 ameliorated motor performance of mice infused with ALS-CSF and it alleviated pathological changes including TDP-43 proteinopathy, neurofilament disorganization, and neuroinflammation. Moreover, CSF infusion experiments were carried out with transgenic mice having neuronal expression of tagged ribosomal protein (hNfL-RFP mice), which allowed immunoprecipitation of neuronal ribosomes for analysis by mass spectrometry of the translational peptide signatures. The results indicate that treatment with IMS-088 prevented many proteomic alterations associated with exposure to ALS-CSF involving pathways related to cytoskeletal changes, inflammation, metabolic dysfunction, mitochondria, UPS, and autophagy dysfunction. The effective disease-modifying effects of this drug in a mouse model based on i.c.v. infusion of ALS-CSF suggest that the NF-κB signaling pathway represents a compelling therapeutic target for sporadic ALS.

Abstract PS06-04: The prognostic and predictive impact of circulating tumour DNA (ctDNA) dynamics in patients with metastatic Triple Negative Breast Cancer (TNBC) on olaparib based therapy: Results from Cohort E of the PlasmaMATCH trial

Abstract Background: Early changes in ctDNA levels, ctDNA dynamics, may help identify which patients are responding to therapy earlier than imaging. Few studies have assessed ctDNA dynamics during PARP inhibitor therapy. Here we report paired baseline and early on treatment ctDNA analysis from cohort E of plasmaMATCH, that recruited patients with TNBC to treatment with olaparib (PARP inhibitor) plus ceralasertib (ATR inhibitor). Methods: The plasmaMATCH trial assessed the ability of ctDNA testing to allocate patients to mutation matched treatment cohorts (A-D). Patients with TNBC, and without mutations matching cohorts B-D, were enrolled on cohort E. Samples were collected for ctDNA analysis pre-treatment at cycle 1-day1 (C1D1) and cycle 2-day 1 (C2D1). A minimum of 14 days of treatment in the first cycle was required for inclusion in this analysis. Samples were sequenced using error-corrected gene targeted panels (Guardant360, or GuardantOMNI, Guardant Health). Circulating DNA ratio (CDR) was calculated as the ratio of C2D1 ctDNA level to C1D1, pre-specified using the weighted mean of variant allele fractions (AF) of clonal mutations at C1D1, excluding variants with AF < 0.3%, and variants in genes frequently mutated in clonal haematopoesis (GNAS, JAK2, IDH1, IDH2 and ATM). The optimal cut-point for predicting progression free survival (PFS) was assessed as the cut-point with the highest Harrell’s C-index. Results: Of the 75 patients that were enrolled into cohort E, 53 patients had samples sent for paired C1D1-C2D1 ctDNA sequencing, 2 failed sequencing, and all 51 (68%) patients had detectable ctDNA at C1D1. The ctDNA analysis set was representative of the overall enrolled population. The optimal ctDNA dynamics C-index cut-point for predicting PFS was 0 (undetectable ctDNA at C2D1). Median PFS with ctDNA CDR >0 (detectable ctDNA at C2D1) was 4.3 months (95% CI 2.4-5.8), and with undetectable ctDNA was 12 months (95% CI 8-NA) (HR 4.02, 95% CI 1.22-13.23, p=0.01). Splitting patients by median CDR was not predictive (HR=0.98; 95%CI: 0.52-1.82, p=0.94). Confirmed objective response rate was 85.7% (42.1-99.6) in patients with undetectable ctDNA at C2D1, and with detectable ctDNA was 11.4% (3.8-24.6) (OR 4.02, 95% CI 1.22-13.23, p=0.01). Of the 7 patients with undetectable ctDNA at C2D1, one had a BRCA2 germline mutation, and all other patients were wildtype for BRCA1/2 mutations in tumour and germline. All patients with undetectable ctDNA and BRCA1/2 wildtype had a confirmed response.In cohorts A-D (mutation targeted therapies, in predominantly ER positive cancer), the optimal ctDNA dynamics C-index CDR cut-point was 0.165 (HR 3.44, 95%CI: 2.06-5.75, p< 0.001), with median CDR cut-point also highly predictive (HR=2.14, 95%CI:1.36-3.36, p=0.001). Undetectable ctDNA was also strongly predictive (HR=4.41; 95%CI: 1.97-9.87, p< 0.001) in cohort A-D.In cohort E, a significant association was found between baseline ctDNA and PFS, with an optimal C-index cutpoint of 6.81% (HR=3.02, 95% CI: 1.39-6.56, p=0.001). Median PFS for baseline ctDNA ≤ 6.81% was 10.2 months (95% CI 3.7-17.2), and for baseline ctDNA >6.81% was 4.4 months (95% CI 2.2-5.5). Conclusions: ‘Clearance’ of ctDNA to become undetectable at C2D1 identified sporadic TNBC patients who benefited from olaparib and ceralasertib. Although ‘clearance’ of ctDNA was associated with good outcome on olaparib plus ceralasertib, median CDR was not predictive of treatment benefit. This contrasts the results of ctDNA dynamic assessment of cohort A-D, where median CDR was highly predictive of treatment benefit. ctDNA dynamic assessment may differ between mutation targeted therapies (cohorts A-D) that induce cell-cycle arrest, and PARP inhibitors (cohort E) that inhibit DNA repair mechanisms. Implementing ctDNA dynamics into clinical trials and care may require distinct analysis for different therapies. Citation Format: Iseult Browne, Javier Pascual, Rosalind Cutts, Belinda Kingston, Sarah Hrebien, Lucy Kilburn, Alex Pearson, Laura Moretti, Andrew Wardley, Iain Macpherson, Richard Baird, Rebecca Roylance, Iris Faull, Kimberly C Banks, Isaac Garcia-Murillas, Judith Bliss, Alistair Ring, Nicholas Turner. The prognostic and predictive impact of circulating tumour DNA (ctDNA) dynamics in patients with metastatic Triple Negative Breast Cancer (TNBC) on olaparib based therapy: Results from Cohort E of the PlasmaMATCH trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS06-04.

A Pilot Study of Detecting RS920778 and RS1899663 Genetic Variants of HOTAIR in A Cohort of Sporadic Breast Cancer Patients in Sri Lanka

Background: Some functional single nucleotide polymorphisms (SNPs) on the HOTAIR gene are potent to elevate breast cancer risk where rs920778 and rs1899663 are associated with the expression and function of the HOTAIR gene, and these SNPs were detected in the current study. Methods: A cohort of nine sporadic cancer patients and five healthy controls were screened for selected HOTAIR variants using direct sequencing. Results: Among patients, seven were with TT genotype, and other 2 with CT genotype for rs920778 C>T while two healthy controls showed TT, another two were with CT, and the remaining with CC genotypes. Among the cohort, the rs1899663 variant was not found in any patients and healthy controls., Conclusion: In the study, the T allele of rs920778 was prominent among the sporadic breast cancer in the studied cohort while only a wild allele (T) of rs1899663 was more prominent in the patients and healthy controls.

Compound heterozygous ABCA12 variants identified in a Chinese patient with congenital ichthyosiform erythroderma: Advancing genotype-phenotype correlations and literature review.

Ichthyosis is a common keratotic skin disease with high clinical, etiological and genetic heterogeneity. There are four types of non-syndromic hereditary ichthyoses, among which autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of recessive Mendelian disorders. ARCI present with different phenotypes and ABCA12 pathogenic variants have been shown to cause complex ARCI phenotypes, including harlequin ichthyosis (HI), lamellar ichthyosis (LI) and congenital ichthyosiform erythroderma (CIE). A sporadic male patient, clinically diagnosed with CIE, was enrolled in this study. Exome sequencing was combined with Sanger sequencing to confirm the diagnosis and identify the pathogenic variants. In silico predictions were made using multiple software programs, and the identified variants were interpreted using the ACMG guidelines. A review of all literature reported ABCA12 variants was performed to explore genotype-phenotype correlations. Compound heterozygous ABCA12 variants [c.5381+1G>A and c.5485G>C (p.Asp1829His)] (NM_173076) were identified. The two variants were not detected in the public database. c.5381+1G>A is predicted to affect ABCA12 mRNA splicing and Asp1829 is highly conserved among various species. In silico analysis suggested that these two variants were responsible for the phenotype of the patient. Genotype-phenotype correlation analysis showed that biallelic truncation variants and/or exon/amino acid deletions in ABCA12 are the most common causes of HI. Biallelic missense variants are most common in LI and CIE. The compound heterozygous ABCA12 variants caused the CIE phenotype observed in the patient. The spectrum of ABCA12 pathogenic variants were broaden. Genotype-phenotype correlation analysis provided detailed evidence which can be used in future prenatal diagnosis and can inform the need for genetic counselling for patients with ABCA12-related ARCIs.

Targeting low levels of MIF expression as a potential therapeutic strategy for ALS

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that macrophage migration inhibitory factor (MIF), whose levels are extremely low in spinal MNs, inhibits mutant SOD1 misfolding and toxicity. In this study, we show that a single peripheral injection of adeno-associated virus (AAV) delivering MIF into adult SOD1G37R mice significantly improves their motor function, delays disease progression, and extends survival. Moreover, MIF treatment reduces neuroinflammation and misfolded SOD1 accumulation, rescues MNs, and corrects dysregulated pathways as observed by proteomics and transcriptomics. Furthermore, we reveal low MIF levels in human induced pluripotent stem cell-derived MNs from familial ALS patients with different genetic mutations, as well as in post mortem tissues of sporadic ALS patients. Our findings indicate that peripheral MIF administration may provide a potential therapeutic mechanism for modulating misfolded SOD1 invivo and disease outcome in ALS patients.

68Ga]DOTATOC PET-derived radiomics to predict genetic background of head and neck paragangliomas: a pilot investigation.

To investigate the [68Ga]DOTATOC PET radiomic profile of head and neck paragangliomas (HNPGLs) and identify radiomic characteristics useful as predictors of succinate dehydrogenase genes (SDHx) pathogenic variants. Sporadic and SDHx HNPGL patients, who underwent [68Ga]DOTATOC PET/CT, were retrospectively included. HNPGLs were analyzed using LIFEx software, and extracted features were harmonized to correct for batch effects and confronted testing for multiple comparison. Stepwise discriminant analysis was conducted to remove redundancy and identify best discriminating features. ROC analysis was used to define optimal cut-offs. Multivariate decision-tree analysis was performed using CHAID method. 34 patients harboring 60 HNPGLs (51 SDHx in 25 patients) were included. Three sporadic and nine SDHx HNPGLs were metastatic. At stepwise discriminant analysis, both GLSZM-Zone Size Non-Uniformity (ZSNU, reflecting tumor heterogeneity) and IB-TLSRE (total lesion somatostatin receptor expression) were independent predictors of genetic status, with 96.4% of lesions and 91.6% of patients correctly classified after cross validation (p < 0.001). Among non-metastatic patients, GLSZM-ZSNU and IB-TLSRE were significantly higher in sporadic than SDHx HNPGLs (p < 0.001). No differences were revealed in metastatic patients. Decision-tree analysis highlights multifocality and IB-TLSRE as useful variables, correctly identifying 6/9 sporadic and 24/25 SDHx patients. Model failed to classify one SDHA and three sporadic patients (2 metastatic). Radiomics features GLSZM-ZSNU and IB-TLSRE appear to reflect HNPGLs SDHx status and tumor behavior (metastatic vs. non-metastatic). If validated, especially IB-TLSRE might represent a simple and time-efficient radiomic index for SDHx variants early screening and prediction of tumor behavior in HNPGL cases.

Modeling sporadic juvenile ALS in iPSC-derived motor neurons explores the pathogenesis of FUSR503fs mutation.

Fused in sarcoma (FUS) mutations represent the most common genetic etiology of juvenile amyotrophic lateral sclerosis (JALS), for which effective treatments are lacking. In a prior report, we identified a novel FUS mutation, c.1509dupA: p. R503fs (FUSR503fs), in a sporadic JALS patient. The physicochemical properties and structure of FUSR503fs protein were analyzed by software: Multi-electrode array (MEA) assay, calcium activity imaging assay and transcriptome analysis were used to explore the pathophysiological mechanism of iPSC derived motor neurons. Structural analysis and predictions regarding physical and chemical properties of this mutation suggest that the reduction of phosphorylation and glycosylation sites, along with alterations in the amino acid sequence, may contribute to abnormal FUS accumulation within the cytoplasm and nucleus of induced pluripotent stem cell- derived motor neurons (MNs). Multi-electrode array and calcium activity imaging indicate diminished spontaneous electrical and calcium activity signals in MNs harboring the FUSR503fs mutation. Transcriptomic analysis reveals upregulation of genes associated with viral infection and downregulation of genes involved in neural function maintenance, such as the ATP6V1C2 gene. Treatment with ropinirole marginally mitigates the electrophysiological decline in FUSR503fs MNs, suggesting the utility of this cell model for mechanistic exploration and drug screening. iPSCs-derived motor neurons from JALS patients are promising tools for drug screening. The pathological changes in motor neurons of FUSR503fs may occur earlier than in other known mutation types that have been reported.

Characterization of spinal hemangioblastomas in patients with and without von Hippel-Lindau, and YAP expression

IntroductionHemangioblastoma (HB) is a benign tumor of the central nervous system, associated with von Hippel-Lindau disease (VHL), or sporadic. The aim of this study was to compare and examine the clinical–pathological profile of patients with spinal hemangioblastoma and YAP expression. MethodsA retrospective, descriptive, comparative study. All patients who underwent surgery for spinal HB between 2016 and 2023 were included. Clinical and radiological data were collected and analyzed. An immunohistochemistry panel including NeuN, neurofilaments (NF), and YAP-1, was performed. ResultsNine patients were studied, six women and three men. Four patients had previously diagnosed VHL. The tumor location included: four cervical (44.44%), two thoracic (22.22%), two pontine with cervical extension (22.22%) and one patient with two lesions, one cervical and one thoracic (11.11%). Non-significant clinical differences were identified between VHL and sporadic patients. Imaging evidenced seven extramedullary and three intramedullary tumors.Histologically, intra-tumoral and perivascular axonal tracts were observed in all cases. One third of the tumors (two with VHL and one sporadic) presented extramedullary hematopoiesis. Seven cases (77.8%) expressed nuclear YAP (three with VHL and four sporadic HBs). The surgical outcome was good and only one patient with VHL undergoing subtotal resection had recurrence. ConclusionsSpinal HBs can be associated with VHL or be sporadic. To the best of our knowledge, this is the first study to describe YAP expression in HB. It is important to investigate the involvement of the Hippo pathway in HBs as a possible therapeutic target.

Enhanced levels of fractalkine and HSP60 in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis patients

Amyotrophic Lateral Sclerosis (ALS) is a multifactorial neurodegenerative disorder with a significant contribution of non-cell autonomous mechanisms to motor neuronal degeneration. Amongst a plethora of molecules, fractalkine (C-X3-C motif chemokine ligand 1), and Heat Shock Protein 60 (HSP60), are key modulators of microglial activation. The contribution of these molecules in Sporadic ALS (SALS) remains unexplored. To investigate this, fractalkine levels were estimated in Cerebrospinal fluid (CSF) of SALS patients (ALS-CSF; n = 44) by Enzyme-linked Immunosorbent Assay (ELISA) and correlated with clinical parameters including disease severity and duration. CSF HSP60 levels were estimated by Western blotting (ALS-CSF; n = 19). Also, CSF levels of Chitotriosidase-1 (CHIT-1), a microglia-specific neuroinflammatory molecule, were measured and its association, if any, with fractalkine and HSP60 was investigated. Both fractalkine and HSP60 levels were significantly elevated in ALS-CSF. Similar to our earlier observation, CHIT-1 levels were also upregulated. Fractalkine showed a moderate negative correlation with the ALS-Functional Rating Scale (ALSFRS) score indicating its significant rise in mild cases which plateaued in cases with high disease severity. However, no obvious correlation was found between fractalkine, HSP60, and CHIT-1. Our study hints that high fractalkine levels in mild cases might be conferring neuroprotection by combating microglial activation and highlights its importance as a novel therapeutic target for SALS. On the other hand, significantly enhanced levels of HSP60, a pro-inflammatory molecule, hint towards its role in accentuating microgliosis, although, it doesn’t act synergistically with CHIT-1. Our study suggests that fractalkine and HSP60 act independently of CHIT-1 to suppress and accentuate neuroinflammation, respectively.

Analysis of Vascular Smooth Muscle Cells from Thoracic Aortic Aneurysms Reveals DNA Damage and Cell Cycle Arrest as Hallmarks in Bicuspid Aortic Valve Patients.

Thoracic aortic aneurysm (TAA) is mainly sporadic and with higher incidence in the presence of a bicuspid aortic valve (BAV) for unknown reasons. The lack of drug therapy to delay TAA progression lies in the limited knowledge of pathophysiology. We aimed to identify the molecular hallmarks that differentiate the aortic dilatation associated with BAV and tricuspid aortic valve (TAV). Aortic vascular smooth muscle cells (VSMCs) isolated from sporadic TAA patients with BAV or TAV were analyzed by mass spectrometry. DNA oxidative damage assay and cell cycle profiling were performed in three independent cohorts supporting proteomics data. The alteration of secreted proteins was confirmed in plasma. Stress phenotype, oxidative stress, and enhanced DNA damage response (increased S-phase arrest and apoptosis) were found in BAV-TAA patients. The increased levels of plasma C1QTNF5, LAMA2, THSB3, and FAP confirm the enhanced stress in BAV-TAA. Plasma FAP and BGN point to an increased inflammatory condition in TAV. The arterial wall of BAV patients shows a limited capacity to counteract drivers of sporadic TAA. The molecular pathways identified support the need of differential molecular diagnosis and therapeutic approaches for BAV and TAV patients, showing specific markers in plasma which may serve to monitor therapy efficacy.

Next-Generation Sequencing in Sporadic Medullary Thyroid Cancer Patients: Mutation Profile and Disease Aggressiveness.

Next-generation sequencing (NGS) analysis of sporadic medullary thyroid carcinoma (sMTC) has led to increased detection of somatic mutations, including RET M918T, which has been considered a negative prognostic indicator. This study aimed to determine the association between clinicopathologic behavior and somatic mutation identified on clinically motivated NGS. In this retrospective cohort study, patients with sMTC who underwent NGS to identify somatic mutations for treatment planning were identified. Clinicopathologic factors, time to distant metastatic disease (DMD), disease-specific survival (DSS), and overall survival (OS) were compared between somatic mutations. Somatic mutations were identified in 191 sMTC tumors, including RET M918T (53.4%), other RET codons (10.5%), RAS (18.3%), somatic RET indels (8.9%), and RET/RAS wild-type (WT) status (8.9%). The median age at diagnosis was 50 years (range, 11-83); 46.1% were female. When comparing patients with RET M918T, RET-Other, and RET WT (which included RAS and RET/RAS WT), there were no differences in sex, TNM category, systemic therapy use, time to DMD, DSS, or OS. On multivariate analysis, older age at diagnosis (HR 1.05, P < .001; HR 1.06, P< .001) and M1 stage at diagnosis (HR 3.17, P = .001; HR 2.98, P = .001) were associated with decreased DSS and OS, respectively, but mutation cohort was not. When comparing RET M918T to RET indels there was no significant difference in time to DMD, DSS, or OS between the groups. Somatic RET mutations do not portend compromised DSS or OS in a cohort of sMTC patients who underwent clinically motivated NGS.

Alterations in Blood Methylome as Potential Epigenetic Biomarker in Sporadic Parkinson's Disease.

To characterize DNA methylation (DNAm) differences between sporadic Parkinson's disease (PD) and healthy control (HC) individuals enrolled in the Parkinson's Progression Markers Initiative (PPMI). Using whole blood, we characterized longitudinal differences in DNAm between sporadic PD patients (n = 196) and HCs (n = 86) enrolled in PPMI. RNA sequencing (RNAseq) was used to conduct gene expression analyses for genes mapped to differentially methylated cytosine-guanine sites (CpGs). At the time of patient enrollment, 5,178 CpGs were differentially methylated (2,683 hypermethylated and 2,495 hypomethylated) in PD compared to HC. Of these, 579 CpGs underwent significant methylation changes over 3 years. Several differentially methylated CpGs were found near the cytochrome P450 family 2 subfamily E member 1 (CYP2E1) gene. Additionally, multiple hypermethylated CpGs were associated with the N-myc downregulated gene family member 4 (NDRG4) gene. RNA-Seq analyses showed 75 differentially expressed genes in PD patients compared to controls. An integrative analysis of both differentially methylated sites and differentially expressed genes revealed 20 genes that exhibited hypomethylation concomitant with overexpression. Additionally, 1 gene, cathepsin H (CTSH), displayed hypermethylation that was associated with its decreased expression. We provide initial evidence of alterations in DNAm in blood of PD patients that may serve as potential epigenetic biomarker of disease. To evaluate the significance of these changes throughout the progression of PD, additional profiling at longer intervals and during the prodromal stages of disease will be necessary. ANN NEUROL 2024;95:1162-1172.

Identification of P21 (CDKN1A) Activated Kinase 4 as a Susceptibility Gene for Familial Non-Medullary Thyroid Carcinoma.

Background: Familial non-medullary thyroid carcinoma (FNMTC) is a genetically predisposed disease with unclear genetic mechanisms. This makes research on susceptibility genes important for the diagnosis and treatment options. Methods: This study included a five-member family affected by papillary thyroid carcinoma. The candidate genes were identified through whole-exome sequencing and Sanger sequencing in family members, other FNMTC patients, and sporadic non-medullary thyroid carcinoma patients. The pathogenicity of the mutation was predicted using in silico tools. Cell phenotype experiments in vitro and models of lung distant metastasis in vivo were conducted to confirm the characteristics of the mutation. Transcriptome sequencing and mechanistic validation were employed to compare the disparities between PAK4 wild-type (WT) and PAK4 mutant (MUT) cell lines. Results: This mutation alters the protein structure, potentially increasing instability by affecting hydrophobicity, intra-molecular hydrogen bonding, and phosphorylation sites. It specifically promotes phosphorylated PAK4 nuclear translocation and expression in thyroid tissue and cell lines. Compared with the WT cells line, PAK4 I417T demonstrates enhanced proliferation, invasiveness, accelerated cell division, and inhibition of cell apoptosis in vitro. In addition, it exhibits a significant propensity for metastasis in vivo. It activates tumor necrosis factor signaling through increased phosphorylation of PAK4, JNK, NFκB, and c-Jun, unlike the WT that activates it via the PAK4-NFκ-MMP9 axis. In addition, PAK4 MUT protein interacts with matrix metalloproteinase (MMP)3 and regulates MMP3 promoter activity, which is not observed in the WT. Conclusions: Our study identified PAK4: c.T1250C: p.I417T as a potential susceptibility gene for FNMTC. The study concludes that the mutant form of PAK4 exhibits oncogenic function, suggesting its potential as a novel diagnostic molecular marker for FNMTC.

TP63 truncating mutation causes increased cell apoptosis and premature ovarian insufficiency by enhanced transcriptional activation of CLCA2

BackgroundPremature ovarian insufficiency (POI) is a severe disorder leading to female infertility. Genetic mutations are important factors causing POI. TP63-truncating mutation has been reported to cause POI by increasing germ cell apoptosis, however what factors mediate this apoptosis remains unclear.MethodsNinety-three patients with POI were recruited from Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Whole-exome sequencing (WES) was performed for each patient. Sanger sequencing was used to confirm potential causative genetic variants. A minigene assay was performed to determine splicing effects of TP63 variants. A TP63-truncating plasmid was constructed. Real-time quantitative PCR, western blot analyses, dual luciferase reporter assays, immunofluorescence staining, and cell apoptosis assays were used to study the underlying mechanism of a TP63-truncating mutation causing POI.ResultsBy WES of 93 sporadic patients with POI, we found a 14-bp deletion covering the splice site in the TP63 gene. A minigene assay demonstrated that the 14-bp deletion variant led to exon 13 skipping during TP63 mRNA splicing, resulting in the generation of a truncated TP63 protein (TP63-mut). Overexpression of TP63-mut accelerated cell apoptosis. Mechanistically, the TP63-mut protein could bind to the promoter region of CLCA2 and activate the transcription of CLCA2 several times compared to that of the TP63 wild-type protein. Silencing CLCA2 using a specific small interfering RNA (siRNA) or inhibiting the Ataxia Telangiectasia Mutated (ATM) pathway using the KU55933 inhibitor attenuated cell apoptosis caused by TP63-mut protein expression.ConclusionOur findings revealed a crucial role for CLCA2 in mediating apoptosis in POI pathogenesis, and suggested that CLCA2 is a potential therapeutic target for POI.

Abstract 6077: Chimeric RNAs are abundantly expressed in precancerous breast tissue of sporadic breast cancer patients

Abstract Background: Immuno-prevention efforts in breast cancer (BC) have been hampered by the lack of known neoantigenic targets that can be used for a cancer prevention vaccine. RNA fusions represent attractive candidates as these are more immunogenic than insertion/deletion mutations and are substantial source of neoantigens. The objective of this study was to characterize chimeric RNAs in precancerous breast tissue. Methodology: Novel RNA fusions were identified by mining the sequence reads from RNA sequencing of 25 Her2 positive, 25 hormone receptor positive (HR) and 25 triple negative (TN) breast tumors and paired histologically normal tissues by Qiagen’s CLC pipeline. Breast samples from women undergoing breast reduction surgery were used as controls to exclude normal tissue associated chimeric mRNAs from analysis. Neoantigen prediction was performed by MHCnuggets. Chimeric mRNAs were validated by PCR. Ribosomal profiling was performed to assess if fusion transcripts are engaged with ribosomes. Results: A median of 36 novel RNA fusions (range 7-877) were identified in the tumor samples. In line with high genomic instability of TNBC subtype of BC, about 1/3 of the TN tumors expressed greater than 300 RNA fusions. In order to identify RNA fusions that are relevant for BC prevention, we investigated the presence of RNA fusions in the paired histologically normal samples as compared to the index tumor. We restricted these analyses to top 20 fusion transcripts that were present across the set of 75 tumor samples and also detected in 1 or more of the TCGA samples. Interestingly, we found that more than 1/3 of the histologically normal samples express at least 1 of the top 20 RNA fusions. When compared to the corresponding tumor, we found up to 50% of the paired normal tissue expressed RNA fusions identified in the tumor, supporting a role for these chimeric mRNA as an early molecular change during breast tumorigenesis. To validate the robustness of the prediction pipeline, PCR validation was performed for the chimeric mRNA with highest prevalence, NSFP1- LRRC37A2 which confirmed the presence of the transcript in 72% (13/18) of the predicted positive patient samples. MHCnuggets pipeline predicted 15 neopeptides from intergenically spliced chimeric NSFP1 [Exon 1-13] - LRRC37A2 [Exon 2-14], ¼ of which we validated to be immunogenic by ELISA. In order to confirm that the translation of the chimeric NSFP1- LRRC37A2 transcript, we investigated their enrichment in polysome enriched fraction obtained from a BC cell line confirmed to express the fusion transcript by PCR. Enrichment of transcripts in the polysomal fraction confirmed that these transcripts are likely to be translated. Conclusions: RNA fusions are frequently present in at risk breast tissue and are the source of substantial number of neoantigens. These RNA fusion- derived neoantigens may provide novel opportunities for developing vaccines for BC prevention. Citation Format: Anjana Bhardwaj, Shiyanth Thevasagayampillai, Sakuni Rankothgedera, Prisha Verma, Micah B. Castillo, Alexander C. Koh, Constance Albarracin, Preethi H. Gunaratne, Isabelle Bedrosian. Chimeric RNAs are abundantly expressed in precancerous breast tissue of sporadic breast cancer patients [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 6077.

Microenvironmental changes in familial adenomatous polyposis during colorectal cancer carcinogenesis

Familial adenomatous polyposis (FAP) is a heritable disease that increases the risk of colorectal cancer (CRC) development because of heterozygous mutations in APC. Little is known about the microenvironment of FAP. Here, single-cell RNA sequencing was performed on matched normal tissues, adenomas, and carcinomas from four patients with FAP. We analyzed the transcriptomes of 56,225 unsorted single cells, revealing the heterogeneity of each cell type, and compared gene expression among tissues. Then we compared the gene expression with that of sporadic CRC. Furthermore, we analyzed specimens of 26 FAP patients and 40 sporadic CRC patients by immunohistochemistry. Immunosuppressiveness of myeloid cells, fibroblasts, and regulatory T cells was upregulated even in the early stages of carcinogenesis. CD8+ T cells became exhausted only in carcinoma, although the cytotoxicity of CD8+ T cells was gradually increased according to the carcinogenic step. When compared with those in the sporadic CRC microenvironment, the composition and function of each cell type in the FAP-derived CRC microenvironment had differences. Our findings indicate that an immunosuppressive microenvironment is constructed from a precancerous stage in FAP.