Real-world experience with circulating tumor DNA in cerebrospinal fluid from patients with central nervous system tumors.

The characterization of genetic alterations in tumor samples has become standard practice for many human cancers to achieve more precise disease classification and guide the selection of targeted therapies. Cerebrospinal fluid (CSF) can serve as a source of tumor DNA in patients with central nervous system (CNS) cancer. We performed comprehensive profiling of CSF circulating tumor DNA (ctDNA) in 711 patients using an FDA-authorized platform (MSK-IMPACT™) in a hospital laboratory. We identified genetic alterations in 489/922 (53.0%) CSF samples with clinically documented CNS tumors. None of 85 CSF samples from patients without CNS tumors had detectable ctDNA. The distribution of clinically actionable somatic alterations was consistent with tumor-type specific alterations across the AACR GENIE cohort. Repeated CSF ctDNA examinations from the same patients identified clonal evolution and emergence of resistance mechanisms. ctDNA detection was associated with shortened overall survival following CSF collection. Next-generation sequencing of CSF, collected through a minimally invasive lumbar puncture in a routine hospital setting, provides clinically actionable cancer genotype information in a large fraction of patients with CNS tumors.

Multi-omic analysis of Huntington's disease reveals a compensatory astrocyte state.

The mechanisms underlying the selective regional vulnerability to neurodegeneration in Huntington's disease (HD) have not been fully defined. To explore the role of astrocytes in this phenomenon, we used single-nucleus and bulk RNAseq, lipidomics, HTT gene CAG repeat-length measurements, and multiplexed immunofluorescence on HD and control post-mortem brains. We identified genes that correlated with CAG repeat length, which were enriched in astrocyte genes, and lipidomic signatures that implicated poly-unsaturated fatty acids in sensitizing neurons to cell death. Because astrocytes play essential roles in lipid metabolism, we explored the heterogeneity of astrocytic states in both protoplasmic and fibrous-like (CD44+) astrocytes. Significantly, one protoplasmic astrocyte state showed high levels of metallothioneins and was correlated with the selective vulnerability of distinct striatal neuronal populations. When modeled in vitro, this state improved the viability of HD-patient-derived spiny projection neurons. Our findings uncover key roles of astrocytic states in protecting against neurodegeneration in HD.

Architecture and dynamics of the abscisic acid gene regulatory network.

The plant hormone abscisic acid (ABA) regulates essential processes in plant development and responsiveness to abiotic and biotic stresses. ABA perception triggers a post-translational signaling cascade that elicits the ABA gene regulatory network (GRN), encompassing hundreds of transcription factors (TFs) and thousands of transcribed genes. To further our knowledge of this GRN, we performed an RNA-seq time series experiment consisting of 14 time points in the 16 h following a one-time ABA treatment of 5-week-old Arabidopsis rosettes. During this time course, ABA rapidly changed transcription levels of 7151 genes, which were partitioned into 44 coexpressed modules that carry out diverse biological functions. We integrated our time-series data with publicly available TF-binding site data, motif data, and RNA-seq data of plants inhibited in translation, and predicted (i) which TFs regulate the different coexpression clusters, (ii) which TFs contribute the most to target gene amplitude, (iii) timing of engagement of different TFs in the ABA GRN, and (iv) hierarchical position of TFs and their targets in the multi-tiered ABA GRN. The ABA GRN was found to be highly interconnected and regulated at different amplitudes and timing by a wide variety of TFs, of which the bZIP family was most prominent, and upregulation of genes encompassed more TFs than downregulation. We validated our network models in silico with additional public TF-binding site data and transcription data of selected TF mutants. Finally, using a drought assay we found that the Trihelix TF GT3a is likely an ABA-induced positive regulator of drought tolerance.

Liquid biopsy-based circulating tumour (ct)DNA analysis of a spectrum of myeloid and lymphoid malignancies yields clinically actionable results.

AIMS: Liquid biopsy (LBx)-based next-generation sequencing (NGS) of circulating tumour DNA (ctDNA) can facilitate molecular profiling of haematopoietic neoplasms (HNs), particularly when tissue-based NGS is infeasible. METHODS AND RESULTS: We studied HN LBx samples tested with FoundationOne Liquid CDx, FoundationOne Liquid, or FoundationACT between July 2016 and March 2022. We identified 271 samples: 89 non-Hodgkin lymphoma (NHL), 43 plasma-cell neoplasm (PCN), 41 histiocytoses, 27 myelodysplastic syndrome (MDS), 25 diffuse large B-cell lymphoma (DLBCL), 22 myeloproliferative neoplasm (MPN), 14 Hodgkin lymphoma (HL), and 10 acute myeloid leukaemia (AML). Among 73.4% with detectable pathogenic alterations, median maximum somatic allele frequency (MSAF) was 16.6%, with AML (36.2%), MDS (19.7%), and MPN (44.5%) having higher MSAFs than DLBCL (3.9%), NHL (8.4%), HL (1.5%), PCN (2.8%), and histiocytoses (1.8%) (P = 0.001). LBx detected characteristic alterations across HNs, including in TP53, KRAS, MYD88, and BTK in NHLs; TP53, KRAS, NRAS, and BRAF in PCNs; IGH in DLBCL; TP53, ATM, and PDCD1LG2 in HL; BRAF and MAP2K1 in histiocytoses; TP53, SF3B1, DNMT3A, TET2, and ASXL1 in MDS; JAK2 in MPNs; and FLT3, IDH2, and NPM1 in AML. Among 24 samples, the positive percent agreement by LBx was 75.7% for variants present in paired buffy coat, marrow, or tissues. Also, 75.0% of pairs exhibited alterations only present on LBx. These were predominantly subclonal (clonal fraction of 3.8%), reflecting the analytical sensitivity of LBx. CONCLUSION: These data demonstrate that LBx can detect relevant genomic alterations across HNs, including at low clonal fractions, suggesting a potential clinical utility for identifying residual or emerging therapy-resistant clones that may be undetectable in site-specific tissue biopsies.

Brain tumors in United States military veterans.

BACKGROUND: Comprehensive analysis of brain tumor incidence and survival in the Veteran population has been lacking. METHODS: Veteran data were obtained from the Veterans Health Administration (VHA) Medical Centers via VHA Corporate Data Warehouse. Brain tumor statistics on the overall US population were generated from the Central Brain Tumor Registry of the US data. Cases were individuals (≥18 years) with a primary brain tumor, diagnosed between 2004 and 2018. The average annual age-adjusted incidence rates (AAIR) and 95% confidence intervals were estimated per 100 000 population and Kaplan-Meier survival curves evaluated overall survival outcomes among Veterans. RESULTS: The Veteran population was primarily white (78%), male (93%), and between 60 and 64 years old (18%). Individuals with a primary brain tumor in the general US population were mainly female (59%) and between 18 and 49 years old (28%). The overall AAIR of primary brain tumors from 2004 to 2018 within the Veterans Affairs cancer registry was 11.6. Nonmalignant tumors were more common than malignant tumors (AAIR:7.19 vs 4.42). The most diagnosed tumors in Veterans were nonmalignant pituitary tumors (AAIR:2.96), nonmalignant meningioma (AAIR:2.62), and glioblastoma (AAIR:1.96). In the Veteran population, survival outcomes became worse with age and were lowest among individuals diagnosed with glioblastoma. CONCLUSIONS: Differences between Veteran and US populations can be broadly attributed to demographic composition differences of these groups. Prior to this, there have been no reports on national-level incidence rates and survival outcomes for Veterans. These data provide vital information that can drive efforts to understand disease burden and improve outcomes for individuals with primary brain tumors.

Multi-OMIC analysis of Huntington disease reveals a neuroprotective astrocyte state.

Huntington disease (HD) is an incurable neurodegenerative disease characterized by neuronal loss and astrogliosis. One hallmark of HD is the selective neuronal vulnerability of striatal medium spiny neurons. To date, the underlying mechanisms of this selective vulnerability have not been fully defined. Here, we employed a multi-omic approach including single nucleus RNAseq (snRNAseq), bulk RNAseq, lipidomics, HTT gene CAG repeat length measurements, and multiplexed immunofluorescence on post-mortem brain tissue from multiple brain regions of HD and control donors. We defined a signature of genes that is driven by CAG repeat length and found it enriched in astrocytic and microglial genes. Moreover, weighted gene correlation network analysis showed loss of connectivity of astrocytic and microglial modules in HD and identified modules that correlated with CAG-repeat length which further implicated inflammatory pathways and metabolism. We performed lipidomic analysis of HD and control brains and identified several lipid species that correlate with HD grade, including ceramides and very long chain fatty acids. Integration of lipidomics and bulk transcriptomics identified a consensus gene signature that correlates with HD grade and HD lipidomic abnormalities and implicated the unfolded protein response pathway. Because astrocytes are critical for brain lipid metabolism and play important roles in regulating inflammation, we analyzed our snRNAseq dataset with an emphasis on astrocyte pathology. We found two main astrocyte types that spanned multiple brain regions; these types correspond to protoplasmic astrocytes, and fibrous-like - CD44-positive, astrocytes. HD pathology was differentially associated with these cell types in a region-specific manner. One protoplasmic astrocyte cluster showed high expression of metallothionein genes, the depletion of this cluster positively correlated with the depletion of vulnerable medium spiny neurons in the caudate nucleus. We confirmed that metallothioneins were increased in cingulate HD astrocytes but were unchanged or even decreased in caudate astrocytes. We combined existing genome-wide association studies (GWAS) with a GWA study conducted on HD patients from the original Venezuelan cohort and identified a single-nucleotide polymorphism in the metallothionein gene locus associated with delayed age of onset. Functional studies found that metallothionein overexpressing astrocytes are better able to buffer glutamate and were neuroprotective of patient-derived directly reprogrammed HD MSNs as well as against rotenone-induced neuronal death in vitro. Finally, we found that metallothionein-overexpressing astrocytes increased the phagocytic activity of microglia in vitro and increased the expression of genes involved in fatty acid binding. Together, we identified an astrocytic phenotype that is regionally-enriched in less vulnerable brain regions that can be leveraged to protect neurons in HD.

Cerebrospinal fluid: A unique source of circulating tumor DNA with broad clinical applications.

Malignancies involving the central nervous system present unique challenges for diagnosis and monitoring due to the difficulties and risks of direct biopsies and the low specificity and/or sensitivity of other techniques for assessment. In recent years, liquid biopsy of the cerebrospinal fluid (CSF) has emerged as a convenient alternative that combines minimal invasiveness with the ability to detect disease-defining or therapeutically actionable genetic alterations from circulating tumor DNA (ctDNA). Since CSF can be obtained by lumbar puncture, or an established ventricular access device at multiple time points, ctDNA analysis enables initial molecular characterization and longitudinal monitoring throughout a patient's disease course, promoting optimization of treatment regimens. This review outlines some of the key aspects of ctDNA from CSF as a highly suitable approach for clinical assessment, the benefits and drawbacks, testing methods, as well as potential future advancements in this field. We anticipate wider adoption of this practice as technologies and pipelines improve and envisage significant improvements for cancer care.

Patterns of TDP-43 Deposition in Brains with LRRK2 G2019S Mutations.

OBJECTIVE: To assess for TDP-43 deposits in brains with and without a LRRK2 G2019S mutation. BACKGROUND: LRRK2 G2019S mutations have been associated with parkinsonism and a wide range of pathological findings. There are no systematic studies examining the frequency and extent of TDP-43 deposits in neuropathological samples from LRRK2 G2019S carriers. METHODS: Twelve brains with LRRK2 G2019S mutations were available for study from the New York Brain Bank at Columbia University; 11 of them had samples available for TDP-43 immunostaining. Clinical, demographic, and pathological data are reported for 11 brains with a LRRK2 G2019S mutation and compared to 11 brains without GBA1 or LRRK2 G2019S mutations with a pathologic diagnosis of Parkinson's disease (PD) or diffuse Lewy body disease. They were frequency matched by age, gender, parkinsonism age of onset, and disease duration. RESULTS: TDP-43 aggregates were present in 73% (n = 8) of brains with a LRRK2 mutation and 18% (n = 2) of brains without a LRRK2 mutation (P = 0.03). In one brain with a LRRK2 mutation, TDP-43 proteinopathy was the primary neuropathological change. CONCLUSIONS: Extranuclear TDP-43 aggregates are observed with greater frequency in LRRK2 G2019S autopsies compared to PD cases without a LRRK2 G2019S mutation. The association between LRRK2 and TDP-43 should be further explored. © 2023 International Parkinson and Movement Disorder Society.

Huntington disease oligodendrocyte maturation deficits revealed by single-nucleus RNAseq are rescued by thiamine-biotin supplementation.

The complexity of affected brain regions and cell types is a challenge for Huntington's disease (HD) treatment. Here we use single nucleus RNA sequencing to investigate molecular pathology in the cortex and striatum from R6/2 mice and human HD post-mortem tissue. We identify cell type-specific and -agnostic signatures suggesting oligodendrocytes (OLs) and oligodendrocyte precursors (OPCs) are arrested in intermediate maturation states. OL-lineage regulators OLIG1 and OLIG2 are negatively correlated with CAG length in human OPCs, and ATACseq analysis of HD mouse NeuN-negative cells shows decreased accessibility regulated by OL maturation genes. The data implicates glucose and lipid metabolism in abnormal cell maturation and identify PRKCE and Thiamine Pyrophosphokinase 1 (TPK1) as central genes. Thiamine/biotin treatment of R6/1 HD mice to compensate for TPK1 dysregulation restores OL maturation and rescues neuronal pathology. Our insights into HD OL pathology spans multiple brain regions and link OL maturation deficits to abnormal thiamine metabolism.

Toward Precision Phenotyping of Multiple Sclerosis.

The classification of multiple sclerosis (MS) has been established by Lublin in 1996 and revised in 2013. The revision includes clinically isolated syndrome, relapsing-remitting, primary progressive and secondary progressive MS, and has added activity (i.e., formation of white matter lesions or clinical relapses) as a qualifier. This allows for the distinction between active and nonactive progression, which has been shown to be of clinical importance. We propose that a logical extension of this classification is the incorporation of additional key pathological processes, such as chronic perilesional inflammation, neuroaxonal degeneration, and remyelination. This will distinguish MS phenotypes that may present as clinically identical but are driven by different combinations of pathological processes. A more precise description of MS phenotypes will improve prognostication and personalized care as well as clinical trial design. Thus, our proposal provides an expanded framework for conceptualizing MS and for guiding development of biomarkers for monitoring activity along the main pathological axes in MS.

The distribution and density of Huntingtin inclusions across the Huntington disease neocortex: regional correlations with Huntingtin repeat expansion independent of pathologic grade.

Huntington disease is characterized by progressive neurodegeneration, especially of the striatum, and the presence of polyglutamine huntingtin (HTT) inclusions. Although HTT inclusions are most abundant in the neocortex, their neocortical distribution and density in relation to the extent of CAG repeat expansion in the HTT gene and striatal pathologic grade have yet to be formally established. We immunohistochemically studied 65 brains with a pathologic diagnosis of Huntington disease to investigate the cortical distributions and densities of HTT inclusions within the calcarine (BA17), precuneus (BA7), motor (BA4) and prefrontal (BA9) cortices; in 39 of these brains, a p62 immunostain was used for comparison. HTT inclusions predominate in the infragranular cortical layers (layers V-VI) and layer III, however, the densities of HTT inclusions across the human cerebral cortex are not uniform but are instead regionally contingent. The density of HTT and p62 inclusions (intranuclear and extranuclear) in layers V-VI increases caudally to rostrally (BA17 < BA7 < BA4 < BA9) with the median burden of HTT inclusions being 38-fold greater in the prefrontal cortex (BA9) than in the calcarine cortex (BA17). Conversely, intranuclear HTT inclusions prevail in the calcarine cortex irrespective of HTT CAG length. Neocortical HTT inclusion density correlates with CAG repeat expansion, but not with the neuropathologic grade of striatal degeneration (Vonsattel grade) or with the duration of clinical disease since motor onset. Extrapolation of these findings suggest that HTT inclusions are at a regionally-contingent, CAG-dependent, density during the advanced stages of HD. The distribution and density of HTT inclusions in HD therefore does not provide a measure of pathologic disease stage but rather infers the degree of pathogenic HTT expansion.

Neurogenetic disorders across the lifespan: from aberrant development to degeneration.

Intellectual disability and autism spectrum disorder (ASD) are common, and genetic testing is increasingly performed in individuals with these diagnoses to inform prognosis, refine management and provide information about recurrence risk in the family. For neurogenetic conditions associated with intellectual disability and ASD, data on natural history in adults are scarce; however, as older adults with these disorders are identified, it is becoming clear that some conditions are associated with both neurodevelopmental problems and neurodegeneration. Moreover, emerging evidence indicates that some neurogenetic conditions associated primarily with neurodegeneration also affect neurodevelopment. In this Perspective, we discuss examples of diseases that have developmental and degenerative overlap. We propose that neurogenetic disorders should be studied continually across the lifespan to understand the roles of the affected genes in brain development and maintenance, and to inform strategies for treatment.

Pituitary corticotroph tumour with adrenocortical cells: A distinct clinicopathologic entity with unique morphology and methylation profile.

We describe a rare TPIT-positive corticotroph PitNET that is admixed with SF1-positive adrenocortical cells. This dimorphous population of cells showed no colocalisation between TPIT and SF1 by immunofluorescence, and an adrenocortical choristoma was favoured. Methylation array analysis revealed a novel methylation profile in relation to other pituitary neoplasms.

Neuropathological Findings in a Case of Parkinsonism and Developmental Delay Associated with a Monoallelic Variant in PLXNA1.

BACKGROUND: PLXNA1 encodes for Plexin-A, a transmembrane protein expressed in the developing nervous system. Mutations in this gene have been associated with developmental delay but have not been previously associated with the development of parkinsonism. OBJECTIVES: To describe the case of a 38-year-old patient with developmental delay who developed parkinsonism later in life. METHODS: Post-mortem exome sequencing was performed with confirmation by Sanger sequencing. Brain autopsy was also performed. RESULTS: Post-mortem exome sequencing on the proband identified a heterozygous predicted nonsense PLXNA1 variant (c.G3361T:p.Glu1121Ter). Pathology demonstrated arhinencephaly with brainstem heterotopia, diffuse Lewy body disease, and frontotemporal lobar dementia-tau. CONCLUSIONS: This case of a patient with developmental delay and parkinsonism with PLXNA1 mutation highlights a need for assessing long-term outcomes of individuals with neurodevelopmental disorders, as well as the need for genetic testing in adults. It also suggests that the link between PLXNA1 and α-synuclein should be explored in the future. © 2021 International Parkinson and Movement Disorder Society.

R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils.

Heterozygous gain-of-kinase function variants in LRRK2 (leucine-rich repeat kinase 2) cause 1-2% of all cases of Parkinson's disease (PD) albeit with incomplete and age-dependent penetrance. All pathogenic LRRK2 mutations reside within the two catalytic domains of LRRK2-either in its kinase domain (e.g. G2019S) with modest effect or its ROC-COR GTPase domain (e.g. R1441G/H) with large effect on LRRK2 kinase activity. We have previously reported assays to interrogate LRRK2 kinase pathway activity in human bio-samples measuring phosphorylation of its endogenous substrate Rab10, that mirrors LRRK2 kinase activation status. Here, we isolated neutrophils from fresh peripheral blood from 101 participants including 42 LRRK2 mutation carriers (21 with the G2019S and 21 with the R1441G mutations), 27 patients with idiopathic PD, and 32 controls. Using a dual approach, LRRK2 dependent Rab10 phosphorylation at Threonine 73 (pRab10Thr73) was measured by quantitative multiplexed immunoblotting for pRab10Thr73/total Rab10 as well as targeted mass-spectrometry for absolute pRab10Thr73 occupancy. We found a significant over fourfold increase in pRab10Thr73 phosphorylation in carriers of the LRRK2 R1441G mutation irrespective of clinical disease status. The effect of the LRRK2 G2019S mutation did not reach statistical significance. Furthermore, we show that LRRK2 phosphorylation at Serine 935 is not a marker for LRRK2 kinase activity in human neutrophils. When analysing pRab10Thr73 phosphorylation in post-mortem brain samples, we observed overall high variability irrespective of clinical and LRRK2 mutation status and attributed this mainly to the adverse effect of the peri- and post-mortem period on the stability of posttranslational modifications such as protein phosphorylation. Overall, in vivo LRRK2 dependent pRab10Thr73 phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation. Additional readouts and/or assays may be needed to increase sensitivity to detect modest LRRK2 kinase activation, as with the LRRK2 G2019S mutation. Our assays could be useful for patient stratification and target engagement studies for LRRK2 kinase inhibitors.