Grade 2, 3 and Dedifferentiated Chondrosarcomas: A Comparative Study of Isocitrate Dehydrogenase-Mutant and Wild-Type Tumors with Implications for Prognosis and Therapy.

BACKGROUND: Grade 2 and 3 and dedifferentiated chondrosarcomas (CS) are frequently associated with isocitrate dehydrogenase (IDH) mutations and often exhibit a poor clinical outcome. Treatment is limited mainly to surgery. Defining IDH status (wild type (WT) and mutant) and the associated transcriptome may prove useful in determining other therapeutic options in these neoplasms. METHODS: Formalin-fixed paraffin-embedded material from 69 primary and recurrent grade 2, 3 and dedifferentiated CS was obtained. DNA sequencing for IDH1 and IDH2 mutations (n = 47) and RNA sequencing via Nextseq 2000 (n = 14) were performed. Differentially expressed genes (DEGs) were identified and used to predict aberrant biological pathways with Ingenuity Pathway Analysis (IPA) software (Qiagen). Gene Set Enrichment Analyses (GSEA) using subsets C3, C5 and C7 were performed. Differentially expressed genes were validated by immunohistochemistry. Outcome analysis was performed using the Wilcoxon test. RESULTS: A set of 69 CS (28 females, 41 males), average age 65, distributed among femur, pelvis, humerus, and chest wall were identified from available clinical material. After further selection based on available IDH status, we evaluated 15 IDH WT and 32 IDH mutant tumors as part of this dataset. Out of 15 IDH WT tumors, 7 involved the chest wall/scapula, while 1 of 32 mutants arose in the scapula. There were far more genes overexpressed in IDH WT tumors compared to IDH mutant tumors. Furthermore, IDH WT and IDH mutant tumors were transcriptomically distinct in the IPA and GSEA, with IDH mutant tumors showing increased activity in methylation pathways and endochondral ossification, while IDH WT tumors showed more activity in normal matrix development pathways. Validation immunohistochemistry demonstrated expression of WT1 and AR in IDH WT tumors, but not in IDH mutants. SATB2 was expressed in IDH mutant tumors and not in WT tumors. Outcome analysis revealed differences in overall survival between mutant and WT tumors (p = 0.04), dedifferentiated mutant and higher-grade (2, 3) mutant tumors (p = 0.03), and dedifferentiated mutant and higher-grade (2, 3) WT tumors (p = 0.03). The longest survival times were observed in patients with higher-grade WT tumors, while patients with dedifferentiated mutant tumors showed the lowest survival. Generally, patients with IDH WT tumors displayed longer survival in both the higher-grade and dedifferentiated groups. CONCLUSIONS: Grade 2, 3 and dedifferentiated chondrosarcomas are further characterized by IDH status, which in turn informs transcriptomic phenotype and overall survival. The transcriptome is distinct depending on IDH status, and implies different treatment targets.

Papillary glioneuronal tumor presenting with low-pressure hydrocephalus from intraventricular spread: Case Report.

Papillary glioneuronal tumors are a rare and typically benign entity with pathological and radiographic complexity. Presentation can mimic other neoplasms, making diagnosis more challenging. The literature to date describes the clinical understanding, diagnostic, therapeutic, and prognostic characteristics of this limited number of patients. In this article, we report an unusual case of a glioneuronal tumor with multifocal presentation, normal pressure hydrocephalus-like symptoms, and large peritumoral parenchymal cysts, which guided the surgical approach. This unusual presentation has not previously been reported and adds valuable information to the clinical recognition and management literature.

Expanding the molecular signatures of malignant ossifying fibromyxoid tumours with two novel gene fusions: PHF1::FOXR1 and PHF1::FOXR2.

AIMS: Ossifying fibromyxoid tumor (OFMT) is a rare enigmatic tumor of uncertain differentiation that can be classified as typical, atypical, and malignant subtypes based on cellularity, nuclear grade, and mitotic activity. The majority of OFMTs, regardless of the risk of malignancy, harbor genetic translocations. We report two malignant OFMTs, including one with evidence of dedifferentiation, with novel genefusions. METHODS AND RESULTS: Case 1 was a 63-year-old male with a dedifferentiated OFMT arising in the right wrist, while case 2 was a 41-year-old male with a malignant OFMT presenting as a posterior mediastinal mass. Case 2 showed multifocal expression with EMA and synaptophysin, while desmin and S100 were absent in both tumors. NGS sequencing studies detected PHF1::FOXR1 and PHF1::FOXR2 gene fusions in cases 1 and 2, respectively. Despite aggressive regimens, both progressed with wide spread metastases resulting in death within six years of diagnosis. CONCLUSIONS: We expand the genetic spectrum of OFMTs with two novel gene fusions, PHF1::FOXR1 and PHF1::FOXR2. These cases confirm the previously reported tendencies for OFMTs with rare variant fusions to demonstrate malignant behavior, unusual morphology, and non-specific immunophenotype.

Germ cell tumors of the central nervous system: A brief review and site-specific considerations.

Germ cell tumors of the central nervous system (GCT-CNS) arise predominantly in midline locations of the CNS and affect young patients in their first to third decades of life. Involvement of the CNS is thought to be a sequelae of residual primordial germ cells with incomplete embryologic migration. Clinically, GCT-CNS present with symptoms of ventricular obstruction or compression of affected brain structures. Histologically, these tumors are analogous to their gonadal and extra-gonadal counterparts. Diagnosis relies heavily on morphology and immunohistochemical findings, and can be complicated by limited tumor sampling. There is currently only a limited role for molecular studies. Treatment of these lesions is made difficult by their involvement of deep and vital brain structures and accurate pathologic diagnosis is essential for appropriate therapy. Diagnosis should involve review of the clinical history, imaging studies, and assessment of serum and cerebrospinal fluid tumor markers. Current therapeutic strategies involving radiation therapy with or without chemotherapy are quite effective, in spite of the locational difficulties that often prevent gross total resection.

Intradural Extramedullary Concurrent Schwannoma and Meningothelial Hyperplasia at C2-C3 Cervical Vertebrae: A Case Report and Review of Literature.

Concomitant schwannomas and benign meningothelial proliferations, including meningothelial hyperplasia or meningioma, rarely occur at the same location outside the setting of neurofibromatosis. Herein, we present a rare case of concurrent schwannoma and benign meningothelial hyperplasia concomitantly occurring in the cervical spine of a 69-year-old male patient with no history of any genetic disorder.

The dominant TP53 hotspot mutation in IDH -mutant astrocytoma, R273C, has distinctive pathologic features and sex-specific prognostic implications.

BACKGROUND: Infiltrative astrocytic tumors with and without isocitrate dehydrogenase (IDH) mutation frequently contain mutations in the TP53 tumor suppressor gene. Disruption of normal p53 protein activity confers neoplastic cells with a number of oncogenic properties and is a common feature of aggressive malignancies. However, the high prevalence of TP53 mutation and its pathogenic role in IDH-mutant (IDHmut) astrocytoma is not well understood. METHODS: We performed a retrospective analysis of molecular and clinical data from patients with IDHmut astrocytoma at the University of Pittsburgh Medical Center between 2015 and 2019 as our initial cohort. We validated and expanded our findings using molecular and clinical data from The Cancer Genome Atlas. RESULTS: We show that the TP53 mutational spectrum in IDHmut astrocytomas is dominated by a single hotspot mutation that codes for the R273C amino acid change. This mutation is not enriched in IDH-wildtype astrocytomas. The high prevalence of TP53 R273C mutation is not readily explained by known mutagenic mechanisms, and TP53 R273C mutant tumors have lower transcriptional levels of proliferation-related genes compared to IDHmut astrocytomas harboring other forms of mutant p53. Despite lower proliferation, TP53 R273C mutant tumors tend to progress more quickly and have a shorter overall survival than those with other TP53 mutations, particularly in male patients. CONCLUSIONS: Our findings suggest that compared to other TP53 mutations, IDHmut astrocytomas may select for TP53 R273C mutations during tumorigenesis. The genotype, sex, and mutation-specific findings are clinically relevant and should prompt further investigation of TP53 R273C.

Three decades of progress from surgery to medical therapy for isolated neuroaxis BRAF V600E-positive Langerhans cell histiocytosis management: illustrative case.

BACKGROUND: "Langerhans cell histiocytosis" (LCH) is a term that encompasses single-system or multisystem disorders traditionally characterized by a proliferation of clonal CD1a+/CD207+ myeloid-derived histiocytes. In most cases of LCH, mitogen-activated protein kinase (MAPK) pathway somatic mutations lead to near universal upregulation of phosphorylated extracellular signal-regulated kinase expression. The clinical manifestations of LCH are numerous, but bone involvement is common. Intracranial lesions, especially as isolated manifestations, are rare. OBSERVATIONS: The authors presented the case of a long-term survivor of exclusive intracranial LCH that manifested with isolated craniofacial bone and intraparenchymal central nervous system recurrences, which were managed with 3 decades of multimodal therapy. The patient was initially diagnosed with LCH at age 2 years, and the authors documented the manifestations of disease and treatment for 36 years. Most of the patient's treatment course occurred before the discovery of BRAF V600E. Treatments initially consisted of chemotherapy, radiosurgery, and open resections for granulomatous LCH lesions. Into young adulthood, the patient had a minimal disease burden but still required additional radiosurgical procedures and open resections. LESSONS: Surgical treatments alleviated the patient's immediate symptoms and allowed for tumor burden control. However, surgical interventions did not cure the underlying, aggressive disease. In the current era, access to systemic MAPK inhibitor therapy for histiocytic lesions may offer improved outcomes.

Homozygous deletion of CDKN2A by fluorescence in situ hybridization is prognostic in grade 4, but not grade 2 or 3, IDH-mutant astrocytomas.

IDH-mutant astrocytomas have a more indolent natural history and better prognosis than their IDH-wild type counterparts, but are still graded according to schemes developed prior to the recognition of this type of neoplasm as a distinct entity. Homozygous deletion of CDKN2A has been proposed as a molecular correlate of aggressive behavior in these tumors, and may be incorporated into future grading systems in an effort to improve prognostic stratification. Fluorescence in situ hybridization (FISH) is a common ancillary testing modality used to assess CDKN2A status, but the specifics of how to best interpret FISH results for prognostication of gliomas have not been clearly defined in the literature. To address this issue, we performed a retrospective analysis of prospectively collected CDKN2A FISH data from 108 primary and 43 recurrent IDH-mutant astrocytomas diagnosed between 2007-2020 at the University of Pittsburgh Medical Center. High level CDKN2A homozygous deletion was rare in primary tumors and was identified more frequently in recurrent tumors. Multivariate Cox Proportional-Hazards analysis demonstrated that histologic grade and CDKN2A status are independent predictors of survival, and the prognostic value of CDKN2A is maximized by applying a threshold of ≥ 30% of tumor cells with homozygous deletion by FISH to define a positive result. At this threshold, CDKN2A deletion significantly stratified survival of histologic grade 4 tumors, but grade 2 and 3 tumors rarely exceeded this cutoff value and did not show worse survival. Lower thresholds identified additional lower grade tumors, but were not prognostically useful. Compared to prior studies, the lack of prognostic significance of CDKN2A homozygous deletion by FISH in grade 2-3 IDH-mutant astrocytomas may reflect differences in cohort populations or technical differences between testing modalities. Definitive criteria for determining CDKN2A homozygous deletion by various methodologies will be critical if this is to be included in future grading schemes.

Multifocal Necrotizing Leukoencephalopathy With Preferential Microglia Toxicity in a Patient Treated With Chimeric Antigen Receptor T-Cells and Review of the Literature.

Neurotoxic side effects of traditional systemic chemotherapy are abundantly described. The introduction of newly developed biologic therapeutics and cellular immune effector therapies has expanded the spectrum of neurotoxicity. Multifocal necrotizing leukoencephalopathy (MNL) is a pathologic condition of unknown etiology that has been observed in patients after prolonged critical illness. We observed a case of MNL in a patient treated with extensive multimodal therapy including chimeric antigen receptor T cells. A month before death, MRI demonstrated signs of inflammation and developing edema in brainstem structures. At autopsy the abnormal MRI regions showed a wave-like loss of microglia with hemorrhagic MNL in regions closest to the brain surface. These findings reiterate the susceptibility of white matter to antineoplastic therapy and suggest new mechanisms of neurotoxicity when traditional chemotherapy is combined with biologic or cellular effector therapy.

Cervical intramedullary spinal cavernoma in setting of unresolved myelopathy: A case report.

BACKGROUND: Spinal cavernous malformations are rare, accounting for approximately 5-12% of all spinal cord vascular lesions. Fortunately, improvements in imaging technologies have made it easier to establish the diagnosis of intramedullary spinal cavernomas (ISCs). CASE DESCRIPTION: Here, we report the case of a 63-year-old male with an >11-year history of left-sided radiculopathy, ataxia, and quadriparesis. Initially, radiographic findings were interpreted as consistent with spondylotic myelopathy with cord signal changes from the C3-C7 levels. The patient underwent a C3-C7 laminectomy/foraminotomy with instrumentation. It was only after several symptomatic recurrences and repeated magnetic resonance images (MRI) that the diagnosis of a ventrally-located intramedullary lesion, concerning for a cavernoma, at the level C6 was established. CONCLUSION: Early and repeated enhanced MR studies may be required to correctly establish the diagnosis and determine the optimal surgical management of ISCs.

Broad Spectrum Mixed Lineage Kinase Type 3 Inhibition and HIV-1 Persistence in Macrophages.

Mixed lineage kinases (MLKs) are a group of serine-threonine kinases that evolved in part to respond to endogenous and exogenous insults that result in oxidative stress and pro-inflammatory responses from innate immune cells. Human immunodeficiency virus type 1 (HIV-1) thrives in these conditions and is associated with the development of associated neurocognitive disorders (HAND). As part of a drug discovery program to identify new therapeutic strategies for HAND, we created a library of broad spectrum MLK inhibitors with drug-like properties. Serendipitously, the lead compound, URMC-099 has proved useful not only in reversing damage to synaptic architecture in models of HAND, but also serves to restore autophagy as a protective response when given in concert with nanoformulated antiretroviral therapy (nanoART) in persistently infected macrophages. These findings are reviewed in the context of MLK3 biology and cellular signaling pathways relevant to new HIV-1 therapies. Graphical abstract.

HIV Tat causes synapse loss in a mouse model of HIV-associated neurocognitive disorder that is independent of the classical complement cascade component C1q.

Microglial activation, increased proinflammatory cytokine production, and a reduction in synaptic density are key pathological features associated with HIV-associated neurocognitive disorders (HAND). Even with combination antiretroviral therapy (cART), more than 50% of HIV-positive individuals experience some type of cognitive impairment. Although viral replication is inhibited by cART, HIV proteins such as Tat are still produced within the nervous system that are neurotoxic, involved in synapse elimination, and provoke enduring neuroinflammation. As complement deposition on synapses followed by microglial engulfment has been shown during normal development and disease to be a mechanism for pruning synapses, we have tested whether complement is required for the loss of synapses that occurs after a cortical Tat injection mouse model of HAND. In Tat-injected animals evaluated 7 or 28 days after injection, levels of early complement pathway components, C1q and C3, are significantly elevated and associated with microgliosis and a loss of synapses. However, C1qa knockout mice have the same level of Tat-induced synapse loss as wild-type (WT) mice, showing that the C1q-initiated classical complement cascade is not driving synapse removal during HIV1 Tat-induced neuroinflammation.

Discovery, synthesis, and characterization of an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3.

Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson's disease and HIV-1 associated neurocognitive disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50 = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC50 = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development.

The new small-molecule mixed-lineage kinase 3 inhibitor URMC-099 is neuroprotective and anti-inflammatory in models of human immunodeficiency virus-associated neurocognitive disorders.

Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) is a significant source of disability in the HIV-infected population. Even with stringent adherence to anti-retroviral therapy, >50% of patients living with HIV-1 will develop HAND (Heaton et al., 2010). Because suppression of viral replication alone is not enough to stop HAND progression, there is a need for an adjunctive neuroprotective therapy in this population. To this end, we have developed a small-molecule brain-penetrant inhibitor with activity against mixed-lineage kinase 3 (MLK3), named URMC-099. MLK3 activation is associated with many of the pathologic hallmarks of HAND (Bodner et al., 2002, 2004; Sui et al., 2006) and therefore represents a prime target for adjunctive therapy based on small-molecule kinase inhibition. Here we demonstrate the anti-inflammatory and neuroprotective effects of URMC-099 in multiple murine and rodent models of HAND. In vitro, URMC-099 treatment reduced inflammatory cytokine production by HIV-1 Tat-exposed microglia and prevented destruction and phagocytosis of cultured neuronal axons by these cells. In vivo, URMC-099 treatment reduced inflammatory cytokine production, protected neuronal architecture, and altered the morphologic and ultrastructural response of microglia to HIV-1 Tat exposure. In conclusion, these data provide compelling in vitro and in vivo evidence to investigate the utility of URMC-099 in other models of HAND with the goal of advancement to an adjunctive therapeutic agent.

LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein.

BACKGROUND: Human Immunodeficiency Virus-1 (HIV-1) associated neurocognitive disorders (HANDs) are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART). While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2) as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. METHODS: We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat) protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i). We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. RESULTS: We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence of Tat-activated microglia, as well as AnnexinV, a phosphatidylserine-binding protein. In addition, LRRK2i decreased brain-specific angiogenesis inhibitor 1 (BAI1) receptor expression on BV-2 cells after Tat-treatment, a key receptor in phosphatidylserine-mediated phagocytosis. CONCLUSION: Taken together, these results implicate LRRK2 as a key player in microglial inflammation and, in particular, in the phagocytosis of neuronal elements. These studies show that LRRK2 kinase inhibition may prove an effective therapeutic strategy for HANDs, as well as other neuroinflammatory conditions.

Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization.

The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.