Langerhans cell histiocytosis (LCH) has a broad spectrum of clinical behaviors; some cases are self-limited, whereas others involve multiple organs and cause significant mortality. Although ...Langerhans cells in LCH are clonal, their benign morphology and their lack (to date) of reported recurrent genomic abnormalities have suggested that LCH may not be a neoplasm. Here, using 2 orthogonal technologies for detecting cancer-associated mutations in formalin-fixed, paraffin-embedded material, we identified the oncogenic BRAF V600E mutation in 35 of 61 archived specimens (57%). TP53 and MET mutations were also observed in one sample each. BRAF V600E tended to appear in younger patients but was not associated with disease site or stage. Langerhans cells stained for phospho-mitogen–activated protein kinase kinase (phospho-MEK) and phospho-extracellular signal-regulated kinase (phospho-ERK) regardless of mutation status. High prevalence, recurrent BRAF mutations in LCH indicate that it is a neoplastic disease that may respond to RAF pathway inhibitors.
Infantile fibrosarcoma and congenital mesoblastic nephroma are tumors of infancy traditionally associated with the ETV6-NTRK3 gene fusion. However, a number of case reports have identified variant ...fusions in these tumors. In order to assess the frequency of variant NTRK3 fusions, and in particular whether the recently identified EML4-NTRK3 fusion is recurrent, 63 archival cases of infantile fibrosarcoma, congenital mesoblastic nephroma, mammary analog secretory carcinoma and secretory breast carcinoma (tumor types that are known to carry recurrent ETV6-NTRK3 fusions) were tested with NTRK3 break-apart FISH, EML4-NTRK3 dual fusion FISH, and targeted RNA sequencing. The EML4-NTRK3 fusion was identified in two cases of infantile fibrosarcoma (one of which was previously described), and in one case of congenital mesoblastic nephroma, demonstrating that the EML4-NTRK3 fusion is a recurrent genetic event in these related tumors. The growing spectrum of gene fusions associated with infantile fibrosarcoma and congenital mesoblastic nephroma along with the recent availability of targeted therapies directed toward inhibition of NTRK signaling argue for alternate testing strategies beyond ETV6 break-apart FISH. The use of either NTRK3 FISH or next-generation sequencing will expand the number of cases in which an oncogenic fusion is identified and facilitate optimal diagnosis and treatment for patients.
Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To ...determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34
lineage
hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.
A sheet of choroid plexus epithelial cells extends into each cerebral ventricle and secretes signaling factors into the CSF. To evaluate whether differences in the CSF proteome across ventricles ...arise, in part, from regional differences in choroid plexus gene expression, we defined the transcriptome of lateral ventricle (telencephalic) versus fourth ventricle (hindbrain) choroid plexus. We find that positional identities of mouse, macaque, and human choroid plexi derive from gene expression domains that parallel their axial tissues of origin. We then show that molecular heterogeneity between telencephalic and hindbrain choroid plexi contributes to region-specific, age-dependent protein secretion in vitro. Transcriptome analysis of FACS-purified choroid plexus epithelial cells also predicts their cell-type-specific secretome. Spatial domains with distinct protein expression profiles were observed within each choroid plexus. We propose that regional differences between choroid plexi contribute to dynamic signaling gradients across the mammalian cerebroventricular system.
Pyogenic granuloma, also called lobular capillary hemangioma, is a condition usually occurring in skin or mucosa and often related to prior local trauma or pregnancy. However, the etiopathogenesis of ...pyogenic granuloma is poorly understood and whether pyogenic granuloma being a reactive process or a tumor is unknown. In an attempt to clarify this issue, we performed genome-wide transcriptional profiling of laser-captured vessels from pyogenic granuloma and from a richly vascularized tissue, placenta, as well as, from proliferative and involutive hemangiomas. Our study identified a gene signature specific to pyogenic granuloma. In the serial analysis of gene expression (SAGE) database, this signature was linked to 'white blood cells monocytes'. It also demonstrated high enrichment for gene ontology terms corresponding to 'vasculature development' and 'regulation of blood pressure'. This signature included genes of the nitric oxide pathway alongside genes related to hypoxia-induced angiogenesis and vascular injury, three conditions biologically interconnected. Finally, one of the genes specifically associated with pyogenic granuloma was FLT4, a tyrosine-kinase receptor related to pathological angiogenesis. All together, these data advocate for pyogenic granuloma to be a reactive lesion resulting from tissue injury, followed by an impaired wound healing response, during which vascular growth is driven by FLT4 and the nitric oxide pathway.
Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae from both extension of the tumors and ...therapeutic interventions that damage the optic chiasm, the pituitary stalk and the hypothalamic area. Using whole-exome sequencing, we identified mutations in CTNNB1 (β-catenin) in nearly all adamantinomatous craniopharyngiomas examined (11/12, 92%) and recurrent mutations in BRAF (resulting in p.Val600Glu) in all papillary craniopharyngiomas (3/3, 100%). Targeted genotyping revealed BRAF p.Val600Glu in 95% of papillary craniopharyngiomas (36 of 39 tumors) and mutation of CTNNB1 in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype, and we detected no other recurrent mutations or genomic aberrations in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms.
Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous ...system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain.
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•CHMP1A is required for progenitor proliferation in developing brain•Loss of CHMP1A decreases sonic hedgehog secretion•Loss of CHMP1A decreases intraluminal vesicles within multivesicular bodies•CHMP1A regulates SHH secretion on the extracellular vesicle subtype ART-EV
Extracellular vesicles (EVs) are essential for cell-to-cell communication in developing brain. Coulter et al. show that the human microcephaly gene CHMP1A is required for neuroprogenitor proliferation through regulation of vesicular secretion of the growth factor sonic hedgehog (SHH). CHMP1A specifically impairs SHH secretion on a distinctive EV subtype, ART-EV.
Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP ...tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues.
Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain ...elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.
•Autosomal recessive loss-of-function mutations in G6b-B (MPIG6B) cause congenital macrothrombocytopenia with focal myelofibrosis.•G6b-B has orthologous physiological functions in human and mice regulating megakaryocyte and platelet production and function.
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Infantile hemangiomas are characterized by rapid capillary growth during the first year of life followed by involution during early childhood. The natural history of these lesions creates a unique ...opportunity to study the changes in gene expression that occur in the vessels of these tumors as they proliferate and regress. Here we use laser capture microdissection and genome-wide transcriptional profiling of vessels from proliferating and involuting hemangiomas to identify differentially expressed genes. Relative to normal placental vessels, proliferating hemangiomas were characterized by increased expression of genes involved in endothelial-pericyte interactions, such as angiopoietin-2 ( ANGPT2 ), jagged-1 ( JAG1 ), and notch-4 ( NOTCH4 ), as well as genes involved in neural and vascular patterning, such as neuropilin-2 ( NETO2 ), a plexin domain containing receptor ( plexinC1 ), and an ephrin receptor ( EPHB3 ). Insulin-like growth factor binding protein-3 ( IGFBP3 ) was down-regulated in proliferating hemangiomas. Involuting hemangiomas were characterized by the expression of chronic inflammatory mediators, such as the chemokine, stromal cell-derived factor-1 ( SDF-1 ), and factors that may attenuate the angiogenic response, such as a member of the Down syndrome critical region ( DSCR ) family. The identification of genes differentially expressed in proliferating and involuting hemangiomas in vivo will contribute to our understanding of this vascular lesion, which remains a leading cause of morbidity in newborn children.
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