Pediatric combined hepatocellular-cholangiocarcinoma (cHCC-CC) with neuroendocrine features: distinguishing genetic alterations detected by chromosomal microarray.

BACKGROUND: Liver tumors exhibiting hepatocellular, cholangiocarcinoma, and neuroendocrine features are extremely rare, with only five cases reported in the literature. CASE PRESENTATION: We present an unusual case of a combined hepatocellular-cholangiocarcinoma (cHCC-CC) with neuroendocrine features in a pediatric patient. A 16-year-old presented with abdominal pain and a 21.0 cm mass in the right hepatic lobe with extension into the left lobe. Histology showed a poorly differentiated tumor with a solid, tubuloglandular, and microcystic architecture. Immunohistochemistry results were negative for hepatic markers, positive for markers of biliary differentiation, and positive for neuroendocrine differentiation. The neoplasm was reviewed at several institutions with differing diagnoses. Single nucleotide polymorphism (SNP) chromosomal microarray (CMA) showed large deletions within chromosomes 6q and 13q in both the hepatocellular-like areas and the cholangiocarcinoma-like areas, with additional large deletions in the cholangiocarcinoma-like areas, supporting origin from hepatocellular carcinoma. The final diagnosis was a cHCC-CC with neuroendocrine features. CONCLUSIONS: Diagnosis of cHCC-CCs relies predominately on histomorphology, as per the 2018 International Consensus Group on the nomenclature of cHCC-CC. These findings in this case support that the pathological classification of these lesions be based on molecular data, which could better direct treatment. Further classification of cHCC-CCs and determination of their clinicopathological relevance will require more interobserver consistency and continued molecular profiling of these lesions.

Immune landscape of a genetically engineered murine model of glioma compared with human glioma.

Novel therapeutic strategies targeting glioblastoma (GBM) often fail in the clinic, partly because preclinical models in which hypotheses are being tested do not recapitulate human disease. To address this challenge, we took advantage of our previously developed spontaneous Qk/Trp53/Pten (QPP) triple-knockout model of human GBM, comparing the immune microenvironment of QPP mice with that of patient-derived tumors to determine whether this model provides opportunity for gaining insights into tumor physiopathology and preclinical evaluation of therapeutic agents. Immune profiling analyses and single-cell sequencing of implanted and spontaneous tumors from QPP mice and from patients with glioma revealed intratumoral immune components that were predominantly myeloid cells (e.g., monocytes, macrophages, and microglia), with minor populations of T, B, and NK cells. When comparing spontaneous and implanted mouse samples, we found more neutrophils and T and NK cells in the implanted model. Neutrophils and T and NK cells were increased in abundance in samples derived from human high-grade glioma compared with those derived from low-grade glioma. Overall, our data demonstrate that our implanted and spontaneous QPP models recapitulate the immunosuppressive myeloid-dominant nature of the tumor microenvironment of human gliomas. Our model provides a suitable tool for investigating the complex immune compartment of gliomas.

A first-in-human Phase I trial of the oral p-STAT3 inhibitor WP1066 in patients with recurrent malignant glioma.

Aim: To ascertain the maximum tolerated dose (MTD)/maximum feasible dose (MFD) of WP1066 and p-STAT3 target engagement within recurrent glioblastoma (GBM) patients. Patients & methods: In a first-in-human open-label, single-center, single-arm 3 + 3 design Phase I clinical trial, eight patients were treated with WP1066 until disease progression or unacceptable toxicities. Results: In the absence of significant toxicity, the MFD was identified to be 8 mg/kg. The most common adverse event was grade 1 nausea and diarrhea in 50% of patients. No treatment-related deaths occurred; 6 of 8 patients died from disease progression and one was lost to follow-up. Of 8 patients with radiographic follow-up, all had progressive disease. The longest response duration exceeded 3.25 months. The median progression-free survival (PFS) time was 2.3 months (95% CI: 1.7 months-NA months), and 6-month PFS (PFS6) rate was 0%. The median overall survival (OS) rate was 25 months (95% CI: 22.5 months-NA months), with an estimated 1-year OS rate of 100%. Pharmacokinetic (PK) data demonstrated that at 8 mg/kg, the T1/2 was 2-3 h with a dose dependent increase in the Cmax. Immune monitoring of the peripheral blood demonstrated that there was p-STAT3 suppression starting at a dose of 1 mg/kg. Conclusion: Immune analyses indicated that WP1066 inhibited systemic immune p-STAT3. WP1066 had an MFD identified at 8 mg/kg which is the target allometric dose based on prior preclinical modeling in combination with radiation therapy and a Phase II study is being planned for newly diagnosed MGMT promoter unmethylated glioblastoma patients.

Central nervous system immune interactome is a function of cancer lineage, tumor microenvironment, and STAT3 expression.

BACKGROUNDImmune cell profiling of primary and metastatic CNS tumors has been focused on the tumor, not the tumor microenvironment (TME), or has been analyzed via biopsies.METHODSEn bloc resections of gliomas (n = 10) and lung metastases (n = 10) were analyzed via tissue segmentation and high-dimension Opal 7-color multiplex imaging. Single-cell RNA analyses were used to infer immune cell functionality.RESULTSWithin gliomas, T cells were localized in the infiltrating edge and perivascular space of tumors, while residing mostly in the stroma of metastatic tumors. CD163+ macrophages were evident throughout the TME of metastatic tumors, whereas in gliomas, CD68+, CD11c+CD68+, and CD11c+CD68+CD163+ cell subtypes were commonly observed. In lung metastases, T cells interacted with CD163+ macrophages as dyads and clusters at the brain-tumor interface and within the tumor itself and as clusters within the necrotic core. In contrast, gliomas typically lacked dyad and cluster interactions, except for T cell CD68+ cell dyads within the tumor. Analysis of transcriptomic data in glioblastomas revealed that innate immune cells expressed both proinflammatory and immunosuppressive gene signatures.CONCLUSIONOur results show that immunosuppressive macrophages are abundant within the TME and that the immune cell interactome between cancer lineages is distinct. Further, these data provide information for evaluating the role of different immune cell populations in brain tumor growth and therapeutic responses.FUNDINGThis study was supported by the NIH (NS120547), a Developmental research project award (P50CA221747), ReMission Alliance, institutional funding from Northwestern University and the Lurie Comprehensive Cancer Center, and gifts from the Mosky family and Perry McKay. Performed in the Flow Cytometry & Cellular Imaging Core Facility at MD Anderson Cancer Center, this study received support in part from the NIH (CA016672) and the National Cancer Institute (NCI) Research Specialist award 1 (R50 CA243707). Additional support was provided by CCSG Bioinformatics Shared Resource 5 (P30 CA046592), a gift from Agilent Technologies, a Research Scholar Grant from the American Cancer Society (RSG-16-005-01), a Precision Health Investigator Award from University of Michigan (U-M) Precision Health, the NCI (R37-CA214955), startup institutional research funds from U-M, and a Biomedical Informatics & Data Science Training Grant (T32GM141746).

Targeting the αv integrin/TGF-ß axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-ß activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-ß signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-ß axis as a potentially useful therapeutic target in GBM.

ERK1/2 phosphorylation predicts survival following anti-PD-1 immunotherapy in recurrent glioblastoma.

Only a subset of recurrent glioblastoma (rGBM) responds to anti-PD-1 immunotherapy. Previously, we reported enrichment of BRAF/PTPN11 mutations in 30% of rGBM that responded to PD-1 blockade. Given that BRAF and PTPN11 promote MAPK/ERK signaling, we investigated whether activation of this pathway is associated with response to PD-1 inhibitors in rGBM, including patients that do not harbor BRAF/PTPN11 mutations. Here we show that immunohistochemistry for ERK1/2 phosphorylation (p-ERK), a marker of MAPK/ERK pathway activation, is predictive of overall survival following adjuvant PD-1 blockade in two independent rGBM patient cohorts. Single-cell RNA-sequencing and multiplex immunofluorescence analyses revealed that p-ERK was mainly localized in tumor cells and that high-p-ERK GBMs contained tumor-infiltrating myeloid cells and microglia with elevated expression of MHC class II and associated genes. These findings indicate that ERK1/2 activation in rGBM is predictive of response to PD-1 blockade and is associated with a distinct myeloid cell phenotype.

Radiation with STAT3 Blockade Triggers Dendritic Cell-T cell Interactions in the Glioma Microenvironment and Therapeutic Efficacy.

PURPOSE: Patients with central nervous system (CNS) tumors are typically treated with radiotherapy, but this is not curative and results in the upregulation of phosphorylated STAT3 (p-STAT3), which drives invasion, angiogenesis, and immune suppression. Therefore, we investigated the combined effect of an inhibitor of STAT3 and whole-brain radiotherapy (WBRT) in a murine model of glioma. EXPERIMENTAL DESIGN: C57BL/6 mice underwent intracerebral implantation of GL261 glioma cells, WBRT, and treatment with WP1066, a blood-brain barrier-penetrant inhibitor of the STAT3 pathway, or the two in combination. The role of the immune system was evaluated using tumor rechallenge strategies, immune-incompetent backgrounds, immunofluorescence, immune phenotyping of tumor-infiltrating immune cells (via flow cytometry), and NanoString gene expression analysis of 770 immune-related genes from immune cells, including those directly isolated from the tumor microenvironment. RESULTS: The combination of WP1066 and WBRT resulted in long-term survivors and enhanced median survival time relative to monotherapy in the GL261 glioma model (combination vs. control P < 0.0001). Immunologic memory appeared to be induced, because mice were protected during subsequent tumor rechallenge. The therapeutic effect of the combination was completely lost in immune-incompetent animals. NanoString analysis and immunofluorescence revealed immunologic reprograming in the CNS tumor microenvironment specifically affecting dendritic cell antigen presentation and T-cell effector functions. CONCLUSIONS: This study indicates that the combination of STAT3 inhibition and WBRT enhances the therapeutic effect against gliomas in the CNS by inducing dendritic cell and T-cell interactions in the CNS tumor.

Sugammadex-Associated Hypotension, Bradycardia, Asystole, and Death.

On December 16, 2015, the Food and Drug Administration (FDA) in the United States approved sugammadex (Bridion, Merck and Co), a modified gamma-cyclodextrin, to be used as a reversal agent. It is a first and unique selective nondepolarizing steroidal muscle relaxant (NDSMR) binding agent with a great affinity for rocuronium and vecuronium. However, there have been several recently published case reports of bradycardia and asystole immediately after sugammadex administration for the reversal. This report presents a case of sugammadex administration followed by rapidly progressing bradycardia leading to asystole and subsequent death. The family has provided the written consent to share this case report.

Verteporfin Inhibits PD-L1 through Autophagy and the STAT1-IRF1-TRIM28 Signaling Axis, Exerting Antitumor Efficacy.

Programmed cell death 1 ligand 1 (PD-L1) is a key driver of tumor-mediated immune suppression, and targeting it with antibodies can induce therapeutic responses. Given the costs and associated toxicity of PD-L1 blockade, alternative therapeutic strategies are needed. Using reverse-phase protein arrays to assess drugs in use or likely to enter trials, we performed a candidate drug screen for inhibitors of PD-L1 expression and identified verteporfin as a possible small-molecule inhibitor. Verteporfin suppressed basal and IFN-induced PD-L1 expression in vitro and in vivo through Golgi-related autophagy and disruption of the STAT1-IRF1-TRIM28 signaling cascade, but did not affect the proinflammatory CIITA-MHC II cascade. Within the tumor microenvironment, verteporfin inhibited PD-L1 expression, which associated with enhanced T-lymphocyte infiltration. Inhibition of chromatin-associated enzyme PARP1 induced PD-L1 expression in high endothelial venules (HEV) in tumors and, when combined with verteporfin, enhanced therapeutic efficacy. Thus, verteporfin effectively targets PD-L1 through transcriptional and posttranslational mechanisms, representing an alternative therapeutic strategy for targeting PD-L1.

Glioblastoma-mediated Immune Dysfunction Limits CMV-specific T Cells and Therapeutic Responses: Results from a Phase I/II Trial.

PURPOSE: Cytomegalovirus (CMV) antigens occur in glioblastoma but not in normal brains, making them desirable immunologic targets. PATIENTS AND METHODS: Highly functional autologous polyclonal CMV pp65-specific T cells from patients with glioblastoma were numerically expanded under good manufacturing practice compliant conditions and administered after 3 weeks of lymphodepleting dose-dense temozolomide (100 mg/m2) treatment. The phase I component used a 3+3 design, ascending through four dose levels (5 × 106-1 × 108 cells). Treatment occurred every 6 weeks for four cycles. In vivo persistence and effector function of CMV-specific T cells was determined by dextramer staining and multiparameter flow cytometry in serially sampled peripheral blood and in the tumor microenvironment. RESULTS: We screened 65 patients; 41 were seropositive for CMV; 25 underwent leukapheresis; and 20 completed ≥1 cycle. No dose-limiting toxicities were observed. Radiographic response was complete in 1 patient, partial in 2. Median progression-free survival (PFS) time was 1.3 months [95% confidence interval (CI), 0-8.3 months]; 6-month PFS was 19% (95% CI, 7%-52%); and median overall survival time was 12 months (95% CI, 6 months to not reached). Repeated infusions of CMV-T cells paralleled significant increases in circulating CMV+ CD8+ T cells, but cytokine production showing effector activity was suppressed, especially from T cells obtained directly from glioblastomas. CONCLUSIONS: Adoptive infusion of CMV-specific T cells after lymphodepletion with dose-dense temozolomide was well tolerated. But apparently CMV seropositivity does not guarantee tumor susceptibility to CMV-specific T cells, suggesting heterogeneity in CMV antigen expression. Moreover, effector function of these T cells was attenuated, indicating a requirement for further T-cell modulation to prevent their dysfunction before conducting large-scale clinical studies.

Comparative Molecular Life History of Spontaneous Canine and Human Gliomas.

Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.

Immune biology of glioma-associated macrophages and microglia: functional and therapeutic implications.

CNS immune defenses are marshaled and dominated by brain resident macrophages and microglia, which are the innate immune sentinels and frontline host immune barriers against various pathogenic insults. These myeloid lineage cells are the predominant immune population in gliomas and can constitute up to 30-50% of the total cellular composition. Parenchymal microglial cells and recruited monocyte-derived macrophages from the periphery exhibit disease-specific phenotypic characteristics with spatial and temporal distinctions and are heterogeneous subpopulations based on their molecular signatures. A preponderance of myeloid over lymphoid lineage cells during CNS inflammation, including gliomas, is a contrasting feature of brain immunity relative to peripheral immunity. Herein we discuss glioma-associated macrophage and microglia immune biology in the context of their identity, molecular drivers of recruitment, nomenclature and functional paradoxes, therapeutic reprogramming and polarization strategies, relevant challenges, and our perspectives on therapeutic modulation.

Genetic and immune profiling for potential therapeutic targets in adult human craniopharyngioma.

Craniopharyngioma is a rare tumor in adults. Although histologically benign, it can be locally aggressive and may require additional therapeutic modalities to surgical resection. Analyses including next generation sequencing, chromogenic and in situ hybridization, immunohistochemistry, and gene amplification were used to profile craniopharyngiomas (n=6) for frequently altered therapeutic targets. Four of six patients had the BRAF V600E missense mutation, frequent in the papillary craniopharyngioma subtype. One patient had a missense mutation in the WNT pathway, specifically CTNNB1, often associated with the adamantinomatous subtype. Craniopharyngiomas lacked microsatellite instability, had low tumor mutational burden, but did express PD-L1 protein, indicating potential therapeutic value for immune checkpoint inhibition. We identified mutations not previously described, including an E318K missense mutation in the MITF gene, an R1407 frameshift in the SETD2 gene of the PIK3CA pathway, R462H in the NF2 gene, and a I463V mutation in TSC2. Two patients testing positive for EGFR expression were negative for the EGFRvIII variant. Herein, we identified several alterations such as those in BRAF V600E and PD-L1, which may be considered as targets for combination therapy of residual craniopharygiomas.

Atypical Presentation of Parietal Convexity Dural-Based Cavernous Hemangioma: A Case Report and Review of Literature.

BACKGROUND: Dural presentation of a cavernous hemangioma is a rare occurrence. Classically an intraparenchymal lesion with varying symptomatology including mostly headaches, seizures, and neurologic deficits depending on its location, a few cases have been reported along the convexity of the brain, even less eroding the calvaria, with none occasioning abnormal movements as the initial presentation. CASE DESCRIPTION: This is a case of a 67-year-old male who presented to the clinic for atypical progressive choreiform movements of the right side of his body and a soft subgaleal mass. Radiographic imaging showed a parietal tumor with intradural and extradural invasion mimicking a meningioma, which does not provide, given its location, a clear explanation of the symptoms. A craniotomy was performed on 29 January, 2018, during which the tumor was resected along with the invaded dura mater and calvaria. CONCLUSIONS: A pathology report was positive for cavernous hemangioma, an unexpected diagnosis based on the tumor's characteristics. After surgery, the patient recovered completely without residual symptoms, suggesting a compressive mass effect causing the atypical movement disorders.