Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children <4 years old; a majority arose in the cerebrum but 24 % (13/54) of tumors had extra-cerebral origins. Notably, group 1 CNS-PNETs encompassed several histologic classes including embryonal tumor with abundant neuropil and true rosettes (ETANTR), medulloepithelioma, ependymoblastoma and CNS-PNETs with variable differentiation. Strikingly, gene expression and methylation profiling analyses revealed a common molecular signature enriched for primitive neural features, high LIN28/LIN28B and DNMT3B expression for all group 1 CNS-PNETs regardless of location or tumor histology. Our collective findings suggest that current known histologic categories of CNS-PNETs which include ETANTRs, medulloepitheliomas, ependymoblastomas in various CNS locations, comprise a common molecular and diagnostic entity and identify inhibitors of the LIN28/let7/PI3K/mTOR axis and DNMT3B as promising therapeutics for this distinct histogenetic entity.

Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known.

We describe the clinical, laboratory, and radiographic characteristics of 15 cases of eastern equine encephalitis in children during 1970-2010. The most common clinical and laboratory features were fever, headache, seizures, peripheral leukocytosis, and cerebrospinal fluid neutrophilic pleocytosis. Radiographic lesions were found in the basal ganglia, thalami, and cerebral cortex. Clinical outcomes included severe neurologic deficits in 5 (33%) patients, death of 4 (27%), full recovery of 4 (27%), and mild neurologic deficits in 2 (13%). We identify an association between a short prodrome and an increased risk for death or for severe disease.

Mutations in SETD2 are found in many tumors, including central nervous system (CNS) tumors. Previous work has shown these mutations occur specifically in high grade gliomas of the cerebral hemispheres in pediatric and young adult patients. We investigated SETD2 mutations in a cohort of approximately 640 CNS tumors via next generation sequencing; 23 mutations were detected across 19 primary CNS tumors. Mutations were found in a wide variety of tumors and locations at a broad range of allele frequencies. SETD2 mutations were seen in both low and high grade gliomas as well as non-glial tumors, and occurred in patients greater than 55 years of age, in addition to pediatric and young adult patients. High grade gliomas at first occurrence demonstrated either frameshift/truncating mutations or point mutations at high allele frequencies, whereas recurrent high grade gliomas frequently harbored subclones with point mutations in SETD2 at lower allele frequencies in the setting of higher mutational burdens. Comparison with the TCGA dataset demonstrated consistent findings. Finally, immunohistochemistry showed decreased staining for H3K36me3 in our cohort of SETD2 mutant tumors compared to wildtype controls. Our data further describe the spectrum of tumors in which SETD2 mutations are found and provide a context for interpretation of these mutations in the clinical setting.

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.

Mutations in RYR1 give rise to diverse skeletal muscle phenotypes, ranging from classical central core disease to susceptibility to malignant hyperthermia. Next-generation sequencing has recently shown that RYR1 is implicated in a wide variety of additional myopathies, including centronuclear myopathy. In this work, we established an international cohort of 21 patients from 18 families with autosomal recessive RYR1-related centronuclear myopathy, to better define the clinical, imaging, and histological spectrum of this disorder. Early onset of symptoms with hypotonia, motor developmental delay, proximal muscle weakness, and a stable course were common clinical features in the cohort. Ptosis and/or ophthalmoparesis, facial weakness, thoracic deformities, and spinal involvement were also frequent but variable. A common imaging pattern consisted of selective involvement of the vastus lateralis, adductor magnus, and biceps brachii in comparison to adjacent muscles. In addition to a variable prominence of central nuclei, muscle biopsy from 20 patients showed type 1 fiber predominance and a wide range of intermyofibrillary architecture abnormalities. All families harbored compound heterozygous mutations, most commonly a truncating mutation combined with a missense mutation. This work expands the phenotypic characterization of patients with recessive RYR1-related centronuclear myopathy by highlighting common and variable clinical, histological, and imaging findings in these patients.

Gastrointestinal stromal tumor (GIST) arises from interstitial cells of Cajal (ICC) or their precursors, which are present throughout the gastrointestinal tract. Although gastric GIST is commonly indolent and small intestine GIST more aggressive, a molecular understanding of disease behavior would inform therapy decisions in GIST. Although a core transcription factor (TF) network is conserved across GIST, accessory TFs HAND1 and BARX1 are expressed in a disease state-specific pattern. Here, we characterize two divergent transcriptional programs maintained by HAND1 and BARX1, and evaluate their association with clinical outcomes.

Genomic studies of high-grade/progressive meningiomas have reported a heterogeneous mutation spectrum, identifying few recurrently mutated genes. Most studies have been underpowered to detect genomic subclasses of aggressive meningiomas due to relatively small number of available samples. Here, we present a genomic survey of one of the largest multi-institutional cohorts of high-grade/progressive meningiomas to date.

Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited neurocutaneous disorder caused by inactivating mutations in TSC1 or TSC2, key regulators of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. In the CNS, TSC is characterized by cortical tubers, subependymal nodules and subependymal giant cell astrocytomas (SEGAs). SEGAs may lead to impaired circulation of CSF resulting in hydrocephalus and raised intracranial pressure in patients with TSC. Currently, surgical resection and mTORC1 inhibitors are the recommended treatment options for patients with SEGA. In the present study, high-throughput RNA-sequencing (SEGAs n = 19, periventricular control n = 8) was used in combination with computational approaches to unravel the complexity of SEGA development. We identified 9400 mRNAs and 94 microRNAs differentially expressed in SEGAs compared to control tissue. The SEGA transcriptome profile was enriched for the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation and survival. Analysis at the protein level confirmed that extracellular signal-regulated kinase (ERK) is activated in SEGAs. Subsequently, the inhibition of ERK independently of mTORC1 blockade decreased efficiently the proliferation of primary patient-derived SEGA cultures. Furthermore, we found that LAMTOR1, LAMTOR2, LAMTOR3, LAMTOR4 and LAMTOR5 were overexpressed at both gene and protein levels in SEGA compared to control tissue. Taken together LAMTOR1-5 can form a complex, known as the 'Ragulator' complex, which is known to activate both mTORC1 and MAPK/ERK pathways. Overall, this study shows that the MAPK/ERK pathway could be used as a target for treatment independent of, or in combination with mTORC1 inhibitors for TSC patients. Moreover, our study provides initial evidence of a possible link between the constitutive activated mTORC1 pathway and a secondary driver pathway of tumour growth.

A high tumour mutational burden (hypermutation) is observed in some gliomas1-5; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.

Introduction: Malignant brain tumors in infants less than 12 months of age are extremely rare, and they have poor prognosis. We evaluated genetic characteristics and response rates of infants with congenital brain tumors subjected to high-dose chemotherapy and autologous stem cell transplant after gross total tumor resection. Materials and Methods: In total, 10 infants, aged less than 12 months, were enrolled in this study. The median age was 56 days (range: 1-279 days). Pathological examination demonstrated the following: four anaplastic astrocytomas, two glioblastomas, two central nervous system (CNS) embryonal tumors, not otherwise specified (NOS), and two atypical teratoid/rhabdoid tumors. Results: All patients were exposed to induction chemotherapy regimen, two high-dose chemotherapy courses, and autologous stem cell transplant after maximal surgery. At 1-3-5 years, the global overall survival (OS) was 90, 70, and 70% and the progression-free survival (PFS) was 80-60 and 60%. In all the patients, the copy number variants (CNVs) profile was analyzed using the SNP/CGH array approach. To investigate the clinical relevance of germline SMARCB1 mutation in AT/RT patients, we performed sequence analysis of the coding regions. The two patients with AT/RT were found to have germline SMARCB1 mutations. No BRAF mutations were found, and only NTRK gene fusion was present in one patient. We also have examined the association with OS and PFS and different histological subtypes of infant CNS proving that high-grade astrocytoma has better overall survival than other tumor types (p: 0.007 and p: 0.0590). Conclusion: High-dose chemotherapy regimen represents a valid therapeutic approach for congenital brain tumors with a high rate of response. The molecular analysis has to be analyzed in all infants' brain tumor types. High-grade gliomas are characterized by a better prognosis than other histologies of infant CNS.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by notorious resistance to current therapies attributed to inherent tumor heterogeneity and highly desmoplastic and immunosuppressive tumor microenvironment (TME). Unique proline isomerase Pin1 regulates multiple cancer pathways, but its role in the TME and cancer immunotherapy is unknown. Here, we find that Pin1 is overexpressed both in cancer cells and cancer-associated fibroblasts (CAFs) and correlates with poor survival in PDAC patients. Targeting Pin1 using clinically available drugs induces complete elimination or sustained remissions of aggressive PDAC by synergizing with anti-PD-1 and gemcitabine in diverse model systems. Mechanistically, Pin1 drives the desmoplastic and immunosuppressive TME by acting on CAFs and induces lysosomal degradation of the PD-1 ligand PD-L1 and the gemcitabine transporter ENT1 in cancer cells, besides activating multiple cancer pathways. Thus, Pin1 inhibition simultaneously blocks multiple cancer pathways, disrupts the desmoplastic and immunosuppressive TME, and upregulates PD-L1 and ENT1, rendering PDAC eradicable by immunochemotherapy.

CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1+ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1+ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6+ TCF1+ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6+ TCF1+ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.

To define the transcriptomic changes responsible for the histologic alterations in skeletal muscle and their progression in collagen VI-related muscular dystrophy (COL6-RD).

Optimal resection of breast tumors requires removing cancer with a rim of normal tissue while preserving uninvolved regions of the breast. Surgical and pathological techniques that permit rapid molecular characterization of tissue could facilitate such resections. Mass spectrometry (MS) is increasingly used in the research setting to detect and classify tumors and has the potential to detect cancer at surgical margins. Here, we describe the ex vivo intraoperative clinical application of MS using a liquid micro-junction surface sample probe (LMJ-SSP) to assess breast cancer margins. In a midpoint analysis of a registered clinical trial, surgical specimens from 21 women with treatment naïve invasive breast cancer were prospectively collected and analyzed at the time of surgery with subsequent histopathological determination. Normal and tumor breast specimens from the lumpectomy resected by the surgeon were smeared onto glass slides for rapid analysis. Lipidomic profiles were acquired from these specimens using LMJ-SSP MS in negative ionization mode within the operating suite and post-surgery analysis of the data revealed five candidate ions separating tumor from healthy tissue in this limited dataset. More data is required before considering the ions as candidate markers. Here, we present an application of ambient MS within the operating room to analyze breast cancer tissue and surgical margins. Lessons learned from these initial promising studies are being used to further evaluate the five candidate biomarkers and to further refine and optimize intraoperative MS as a tool for surgical guidance in breast cancer.

Proliferation is a fundamental trait of cancer cells, but its properties and spatial organization in tumours are poorly characterized. Here we use highly multiplexed tissue imaging to perform single-cell quantification of cell cycle regulators and then develop robust, multivariate, proliferation metrics. Across diverse cancers, proliferative architecture is organized at two spatial scales: large domains, and smaller niches enriched for specific immune lineages. Some tumour cells express cell cycle regulators in the (canonical) patterns expected of freely growing cells, a phenomenon we refer to as 'cell cycle coherence'. By contrast, the cell cycles of other tumour cell populations are skewed towards specific phases or exhibit non-canonical (incoherent) marker combinations. Coherence varies across space, with changes in oncogene activity and therapeutic intervention, and is associated with aggressive tumour behaviour. Thus, multivariate measures from high-plex tissue images capture clinically significant features of cancer proliferation, a fundamental step in enabling more precise use of anti-cancer therapies.

Precision medicine is critically dependent on better methods for diagnosing and staging disease and predicting drug response. Histopathology using hematoxylin and eosin (H&E)-stained tissue (not genomics) remains the primary diagnostic method in cancer. Recently developed highly multiplexed tissue imaging methods promise to enhance research studies and clinical practice with precise, spatially resolved single-cell data. Here, we describe the 'Orion' platform for collecting H&E and high-plex immunofluorescence images from the same cells in a whole-slide format suitable for diagnosis. Using a retrospective cohort of 74 colorectal cancer resections, we show that immunofluorescence and H&E images provide human experts and machine learning algorithms with complementary information that can be used to generate interpretable, multiplexed image-based models predictive of progression-free survival. Combining models of immune infiltration and tumor-intrinsic features achieves a 10- to 20-fold discrimination between rapid and slow (or no) progression, demonstrating the ability of multimodal tissue imaging to generate high-performance biomarkers.

Homeostasis of normal tissues and the emergence of diseases such as cancer are controlled by changes in the proportions and states of diverse cell types, cell-cell interactions, and acellular components of the tissue microenvironment1. Spatial omics using highly multiplexed tissue profiling2 makes it possible to study these processes in situ, usually on thin, 4-5 micron thick sections (the standard histopathology format)3. Microscopy-based tissue imaging is commonly performed at a resolution sufficient to determine cell types but not detect the subtle morphological features associated with cytoskeletal reorganisation, juxtracrine signalling, or membrane trafficking4. Here we introduce a 3D imaging approach using existing instruments and reagents that is able to characterize a wide variety of organelles and structures at sub-micron scale while simultaneously quantifying millimetre-scale spatial features. We perform high-resolution 54-plex cyclic immunofluorescence (CyCIF) imaging3 on sections of primary human melanoma thick enough (30-40 microns) to fully encompass two or more layers of intact cells. In pre-invasive melanoma in situ5, 3D imaging of entire cell volumes showed that transformed melanocytic cells are plastic in state and participate in tightly localised niches of interferon signalling near sites of initial invasion into the underlying dermis. Below this layer, immune cells engaged in an unexpectedly diverse array of membrane-membrane interactions as well as looser "neighbourhood" associations6. These data provide new insight into the transitions occurring during early tumour formation and demonstrate the potential for phenotyping tissues at a level of detail previously restricted to cultured cells and organoids.

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.