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Background: Glioblastoma mortality is driven by tumour progression or recurrence despite administering a therapeutic arsenal consisting of surgical resection, radiation, and alkylating chemotherapy. The genetic changes underlying tumour progression and chemotherapy resistance are poorly understood. Methods: In this study, we sought to define the relationship between EGFR amplification status, EGFR mRNA expression, and EGFR pathway activity. We compared RNA-sequencing data from matched primary and recurrent tumour samples (n = 40 patients, 20 with EGFR amplification). Results: In the setting of glioblastoma recurrence, the EGFR pathway was overexpressed regardless of EGFR-amplification status, suggesting a common genomic endpoint in recurrent glioblastoma, although EGFR amplification did associate with higher EGFR mRNA expression. Three of forty patients in the study cohort had EGFR-amplified tumours and received targeted EGFR therapy. Their molecular subtypes and clinical outcomes did not significantly differ from patients who received conventional chemotherapy. Conclusion: Our findings suggest that while the EGFR amplification may confer a unique molecular profile in primary glioblastoma, pathway analysis reveals upregulation of the EGFR pathway in recurrence, regardless of amplification status. As such, the EGFR pathway may be a key mediator of glioblastoma progression.

High-grade astrocytoma with piloid features (HGAP) is a recently recognized glioma type whose classification is dependent on its global epigenetic signature. HGAP is characterized by alterations in the mitogen-activated protein kinase (MAPK) pathway, often co-occurring with CDKN2A/B homozygous deletion and/or ATRX mutation. Experience with HGAP is limited and to better understand this tumor type, we evaluated an expanded cohort of patients (n = 144) with these tumors, as defined by DNA methylation array testing, with a subset additionally evaluated by next-generation sequencing (NGS). Among evaluable cases, we confirmed the high prevalence CDKN2A/B homozygous deletion, and/or ATRX mutations/loss in this tumor type, along with a subset showing NF1 alterations. Five of 93 (5.4%) cases sequenced harbored TP53 mutations and RNA fusion analysis identified a single tumor containing an NTRK2 gene fusion, neither of which have been previously reported in HGAP. Clustering analysis revealed the presence of three distinct HGAP subtypes (or groups = g) based on whole-genome DNA methylation patterns, which we provisionally designated as gNF1 (n = 18), g1 (n = 72), and g2 (n = 54) (median ages 43.5 years, 47 years, and 32 years, respectively). Subtype gNF1 is notable for enrichment with patients with Neurofibromatosis Type 1 (33.3%, p = 0.0008), confinement to the posterior fossa, hypermethylation in the NF1 enhancer region, a trend towards decreased progression-free survival (p = 0.0579), RNA processing pathway dysregulation, and elevated non-neoplastic glia and neuron cell content (p < 0.0001 and p < 0.0001, respectively). Overall, our expanded cohort broadens the genetic, epigenetic, and clinical phenotype of HGAP and provides evidence for distinct epigenetic subtypes in this tumor type.

PURPOSE To report a case of vitreous seeding in a medium-sized choroidal melanoma and review the literature. METHODS Observational case report and review of literature for pathogenesis, role of vitreous biopsy, and treatment outcomes. RESULTS A case of 57-year-old man diagnosed with vitreous seeding in the left eye 1 year after episcleral brachytherapy for medium-sized choroidal melanoma. The patient was initially diagnosed to have subretinal and vitreous hemorrhage due to rupture of a retinal artery macroaneurysm for which focal laser and intravitreal antivascular endothelial growth factor injections were administered. Over the next 9 months, the vitreous hemorrhage cleared and choroidal melanoma with retinal invasion became evident. One year after brachytherapy, the primary tumor regressed with resolution of surrounding subretinal fluid and hemorrhage. However, gradual decline in the visual acuity from 20/50 to 20/500 with increase of pigmented debris over the retinal surface and in the vitreous cavity was noted. A vitreous biopsy confirmed the presence of viable melanoma cells (epithelioid type), and the eye was enucleated. Histopathology showed microscopic persistence of primary tumor with diffuse vitreous seeding. CONCLUSION Vitreous seeding of choroidal melanoma poses a diagnostic and management challenge.

Uveal melanoma (UM) and renal cell carcinoma (RCC) can occur sporadically and as a manifestation of BAP1 tumor predisposition syndrome. We aimed to understand the prevalence of germ line BAP1 pathogenic variants in patients with UM and RCC. We reviewed patients managed at Cleveland Clinic between November 2003 and November 2019 who were diagnosed with UM and RCC. Charts were reviewed for demographic and cancer-related characteristics. RCC samples were tested for BAP1 protein expression using immunohistochemical (IHC) staining, and testing for germ line BAP1 pathogenic variants was performed as part of routine clinical care. Thirteen patients were included in the study. The average age at diagnosis of UM was 61.3 years. Seven patients underwent fine-needle aspiration biopsy for prognostic testing of UM (low risk =5, high risk =2). Twelve patients were treated with plaque radiation therapy, and 3 patients developed metastatic disease requiring systemic therapy. The median time to diagnosis of RCC from time of diagnosis of UM was 0 months. RCC samples were available for 7 patients for BAP1 IHC staining (intact =6, loss =1). All patients underwent nephrectomy (total = 3, partial = 8, unknown =2), and 1 received systemic therapy for metastatic RCC. Six patients underwent germ line BAP1 genetic testing. Of these, 1 patient was heterozygous for a pathogenic variant of BAP1 gene: c.1781-1782delGG, p.Gly594Valfs*48. The overall prevalence of germ line BAP1 pathogenic variants in our study was high (1/6; 17%; 95% CI 0–46%). Patients with UM and RCC should be referred for genetic counseling to discuss genetic testing.

Purpose: To report a case of local metastasis of conjunctival melanoma, which may occur via extravascular migratory metastasis (EVMM), and discuss its clinical relevance in conjunctival melanoma tumor staging and possible management implications. Methods: Retrospective chart review of a single clinical case with clinicopathologic correlation. Results: A 65-year-old male referred due to local recurrence of conjunctival melanoma at the caruncle was successfully treated after two excisional procedures with negative sentinel lymph node biopsies. Forty-eight months after initial presentation, the patient developed a nodular lesion representing local recurrence in the ipsilateral upper tarsal conjunctiva, distant from the primary tumor site. Histopathology showed nodules in the substantia propria in the absence of primary acquired melanosis. The tumor cells were found along the extravascular surface without intralymphatic or intravascular tumor cells consistent with local metastasis. One possible mechanism is angiotropic microsatellitosis leading to local EVMM. Additional neck CT imaging showed no lymphadenopathy. Conclusion: EVMM via angiotropic microsatellitosis is another possible mechanism of noncontiguous local recurrence of conjunctival melanoma. Angiotropic microsatellitosis may represent a high-risk finding possibly related to increased melanoma-related mortality.

Purpose To assess margins and the rate of local recurrence of ocular surface squamous neoplasia after excision with a 2 mm margin and cryotherapy at a single ophthalmic oncology center. Outcome measures (1) Conjunctival margin were assessed as positive, negative, or indeterminate margins. (2) Feasibility of repair without a graft. (3) Local recurrence. Methods Retrospective chart review of histologically proven conjunctival intraepithelial neoplasia and invasive squamous cell carcinoma cases that underwent excision with a 2 mm margin and cryotherapy. Results Eighty cases met inclusion criteria for the quantitative analysis. The margin was positive in six cases (7.5%), four of which were treated with post-op topical immunotherapy/chemotherapy. Of the six positive margin cases, there was one recurrence which occurred in the patient who did not receive post-op topical adjuvant therapy, however resolved after starting topical treatment. Conjunctival repair without use of a graft was feasible in 74 (93%) cases with a mean basal diameter of 6.4 mm. Total number of local recurrence was seen in three cases (4%), which were successfully treated with adjuvant topical treatment (one positive margin case, one indeterminate margin case) or repeat resection followed by episcleral plaque brachytherapy (one negative margin case). Conclusion Excision with 2 mm margin of OSSN is not associated with high rates of positive surgical margins. Even those with positive margins, when treated with adjuvant topical therapy did not develop recurrence. While achieving low rates of local recurrence, the conjunctiva is conserved, thereby minimizing the need for amniotic membrane or free conjunctival grafts for conjunctival repair.

AimTo assess the role of map biopsy in patients with conjunctival primary acquired melanosis (PAM)/melanoma.MethodsRetrospective case series of 400 conjunctival biopsy samples of 51 unique patients in a tertiary referral centre.ResultsEach patient underwent one diagnostic biopsy and several additional map biopsies (range 2–7) providing a total of 400 samples for the analysis (55 diagnostic biopsies, 345 map biopsies). The median age was 63 years old (range 20–88) with women representing 67% of the cases. Histopathological findings were graded as negative for melanosis/normal (grade 0), melanosis without atypia (grade 1), melanosis with mild atypia (grade 2), melanosis with severe atypia (grade 3) or invasive melanoma (grade 4). Clinicopathologic concordance was observed in the majority of the map biopsies (313, 91%) (positive: clinical+/path+ (57,17%), negative: clinical−/path− (256, 74%)). Three discordant samples (clinical−/path+) represented PAM sine pigmento. The histopathological spectrum of atypia was absent (40, 73%) or limited (11, 20%) in the majority of cases with tendency to cluster as low-grade or high-grade atypia. Map biopsy led to the identification of six patients (11%) with severe atypia, requiring topical mitomycin (MMC). Similarly, in 29 cases, periodic observation without topical MMC was recommended. One case of invasive melanoma transformation occurred in the MMC-treated group.ConclusionsMap biopsy enhances overall assessment of the anatomic and pathologic extent, impacting use of adjuvant topical chemotherapy. In absence of map biopsy, it would be impossible to diagnose PAM sine pigmento. Additional corroborative work is needed to validate our observations.

Uveal melanoma (UM) and renal cell carcinoma (RCC) can occur sporadically and as a manifestation of BAP1 tumor predisposition syndrome. We aimed to understand the prevalence of germ line BAP1 pathogenic variants in patients with UM and RCC. We reviewed patients managed at Cleveland Clinic between November 2003 and November 2019 who were diagnosed with UM and RCC. Charts were reviewed for demographic and cancer-related characteristics. RCC samples were tested for BAP1 protein expression using immunohistochemical (IHC) staining, and testing for germ line BAP1 pathogenic variants was performed as part of routine clinical care. Thirteen patients were included in the study. The average age at diagnosis of UM was 61.3 years. Seven patients underwent fine-needle aspiration biopsy for prognostic testing of UM (low risk =5, high risk =2). Twelve patients were treated with plaque radiation therapy, and 3 patients developed metastatic disease requiring systemic therapy. The median time to diagnosis of RCC from time of diagnosis of UM was 0 months. RCC samples were available for 7 patients for BAP1 IHC staining (intact =6, loss =1). All patients underwent nephrectomy (total = 3, partial = 8, unknown =2), and 1 received systemic therapy for metastatic RCC. Six patients underwent germ line BAP1 genetic testing. Of these, 1 patient was heterozygous for a pathogenic variant of BAP1 gene: c.1781-1782delGG, p.Gly594Valfs*48. The overall prevalence of germ line BAP1 pathogenic variants in our study was high (1/6; 17%; 95% CI 0–46%). Patients with UM and RCC should be referred for genetic counseling to discuss genetic testing.

Purpose Ocular adnexal amyloidosis (OAA) may represent localized manifestation of an underlying systemic process. Accurate identification of the amyloid fibrils can guide the systemic evaluation and treatment. The aim of this study was to characterize subtypes of OAA using immunohistochemistry and mass spectrometric analysis and to correlate with ocular involvement and systemic association. Design Retrospective case series. Methods Review of patients with OAA subtyped by immunohistochemistry and mass spectrometric analysis at the Cleveland Clinic from June 1995 to June 2017. Results While immunohistochemistry identified AL amyloid protein in 67% (4/6) of specimens tested, mass spectrometry identified AL amyloid protein in all specimens (10/10). AL lambda was identified in 5 (50%) samples, kappa in 3 (30%), and both kappa and lambda light chains in 2 (20%). The 5 cases of conjunctival amyloidosis were either AL lambda only (3 cases) or both lambda and kappa (2 cases). There were 3 cases that had associated systemic involvement. Two of these had eyelid skin involvement and AL kappa amyloidosis and the other patient had uveal involvement and AL lambda amyloidosis. Conclusions Primary amyloidosis-AL is the most common form diagnosed by mass spectrometric analysis in patients with OAA. Immunohistochemistry is ineffective in the characterization of the amyloid deposits in a significant number of cases. Evaluation to exclude systemic involvement or associated underlying lymphoproliferative disorder is warranted.

World Health Organization (WHO) central nervous system tumor classification represents the primary source of updates on diagnostic classes, grades, and criteria. However, recent and ongoing advances in molecular pathogenesis warrant more rapid integration into clinical practice between WHO updates. To accomplish this, the consortium to inform molecular and practical approaches to CNS tumor taxonomy-not official WHO (cIMPACT-NOW) was established in 2016. Since then, cIMPACT-NOW has convened 3 separate working committees to address classification and grading questions and challenges. This review covers the progress that these working committees have made on their specific topics.

Intravascular T-cell lymphomas are rare, poorly characterized lesions. We discuss the clinical, radiologic and especially the laboratory characteristics of a lesion which presented in a 62-year-old woman with a history of progressive CNS abnormalities. Throughout the course of the disease, radiologic findings consisted mainly of multifocal mixed areas of ischemia and vasogenic edema involving cortical and subcortical regions. A brain biopsy identified an abnormal T-cell population confined to lumens of vessels. These T-cells were abnormal cytotoxic cells, positive for CD3, CD8, and negative for CD2, CD4, CD5, CD7 and CD30. While flow cytometry and immunohistochemistry failed to identify a similar population in the blood or bone marrow, molecular studies showed a clonal T-cell population in both the brain and the bone marrow. No other organs were involved. In spite of aggressive treatment, the patient's medical condition continued to progress and she passed away. In conclusion, this abnormal population of cytotoxic T-cells with intravascular localization probably represents a specific type of T-cell lymphoma with specific clinical, radiologic, molecular and immunophenotypic characteristics.

A 66-year-old man presented with a 6-month history of worsening confusion and panhypopituitarism. MRI showed a 1.7-cm enhancing mass involving the infundibulum and hypothalamus.1 Total body PET scan showed isolated uptake in the hypothalamus (standardized uptake value 25.3) (figure 1). Full body CT, lumbar puncture, and infectious workup were negative. Endoscopic transventricular biopsies were performed. Microscopy revealed gliotic hypothalamus with clusters of cells with elongated grooved nuclei positive for CD1a, Langerin, BRAF ( V600E ), CD68, CD163, and S-100 by immunohistochemistry and negative for Grocott methenamine silver, Gram stain, acid-fast bacilli, Warthin-Starry, herpes simplex virus 1 and 2, Epstein-Barr virus, and β-amyloid, confirming the diagnosis of Langerhans cell histiocytosis (figure 2). The finding of BRAF (V600E) positivity is important diagnostically and as a potential therapeutic target.2

INTRO/OBJECTIVE Glioblastoma (GBM) and MGMT have been reported to have sexual dimorphism. The primary objective of this study was to analyze the impact and association between sex and MGMT status on progression-free survival (PFS) and overall survival (OS) in patients with newly diagnosed GBM. METHODS 582 patients with newly diagnosed GBM who underwent first surgical intervention at a single tertiary care institution between 2012 and 2018 were reviewed. Adults with documented methylated (≥ 12) and un-methylated (≤ 7) MGMT status were included. A Kaplan-Meier and Cox proportional hazard models were used to analyze the association between sex and MGMT status on PFS and OS. RESULTS 464 adult patients (median age 63.4, 36.6% female) had documented MGMT status. Overall rate of MGMT methylated patients was 42.5%, while females were more often methylated than males (52.1% vs 37.4%, p=0.004). MGMT methylated compared to un-methylated females (median: 12.8 vs 7.4 months; 1-yr: 53% vs 27%) had a greater PFS benefit than males (median: 9.6 vs 6.8 months; 1-yr: 44% vs 23%). OS was significantly improved in MGMT methylated compared to un-methylated patients among females (p=0.001) but not among males (p=0.22). Among MGMT methylated patients, females had significantly better OS compared to males (median: 18.7 vs 12.4 months; 2-yr OS: 36.8% vs 24.3%, p=0.03). Although statistically not significant, a similar pattern was observed on PFS (median: 12.8 vs 9.6 months; 1-yr PFS: 52.6% vs 44.4%). Compared to MGMT methylated females, MGMT methylated males had a PFS HR=1.22 (95% CI=0.80 – 1.85, p=0.36), and an OS HR=1.45 (95% CI=1.03 – 2.04, p=0.032). CONCLUSION MGMT methylation is more common in females and methylation had a larger impact on both PFS and OS in females compared to males. These analyses highlight the need to further investigate sex differences that can inform clinical management of GBM.

INTRO/OBJECTIVE Glioblastoma (GBM) and MGMT have been reported to have sexual dimorphism. In multiple studies, including our own population-based cohort analysis, females had higher rates of MGMT methylation and improved methylation-associated progression-free and overall survival outcomes compared to males. MGMT methylation is assessed as a mean of five cysteine-phosphate-guanine (CpG1-5) islands (CpG methylation is highly inversely correlated with MGMT RNA expression). The primary objective of this study was to investigate differences in mean and individual CpG methylation by sex. METHODS 155 patients who underwent first surgical intervention for newly diagnosed GBM at a single tertiary care institution between 2016 and 2018 were reviewed. Of these, 135 patients had available CpG methylation data determined by a clinically validated test using bisulfate conversion followed by PCR and pyrosequencing. MGMT was defined as methylated if the mean of CpG1-5 ≥ 12. The mean of CpG1-5 and each CpG parameter were compared by sex using the Wilcoxon signed-rank test. RESULTS Overall (mean age 62, 34% female, 42% MGMT methylated), the median (IQR) of mean degree of methylation was 4.0% (2–33) and median CpG1-5 ranged from 3.0 to 4.5%. More females (53.3%) were MGMT methylated than males (37.1%). Females had significantly higher rates of mean methylation compared to males (14.0 vs 3.0%, p=0.046). Females also had higher rates of methylation at each CpG island compared to males CpG1(7.0 vs 3.0%, p=0.15), CpG2(8.0 vs 4.0%, p=0.10), CpG3(9.0 vs 4.0%, p=0.23), CpG4(7.0 vs 3.0%, p=0.047), and CpG5(6.0 vs 4.0%, p=0.097). CONCLUSION Females had higher rates of mean methylation and methylation of each CpG island compared to males, although only mean and CpG4 methylation values were statistically significant given the limited sample size. Further investigation with a larger cohort is ongoing to elucidate this dimorphism and establish whether sex-specific methylation cut-offs need to be implemented into clinical practice.

A near-universal phenomenon in glioblastoma is disease recurrence following surgical resection and chemoradiotherapy. Development of biomarkers predictive of therapeutic response to better guide care and inform future targeted therapies is crucial. In this work, a total of 84 glioblastoma surgical specimens involving 44 primary tumors and 40 matched samples at time of re-resection, were characterized utilizing RNA-sequencing. Transcriptomic analysis was carried out with the goal of identifying underlying differences between those patients with prolonged response to standard therapy and delayed time to re-resection. We examined individual gene expression, gene coexpression networks, and well-known gene pathways in this dataset that showed consistent association with time to re-resection in both primary and progressed specimens, independent of tumor molecular subtype. Leveraging this large, well-characterized dataset, and using a novel computational methodology based on a seed-gene approach, we identified a predictive gene signature for therapeutic response. Our analyses revealed a striking degree of heterogeneity among gene expression associated with response to standard therapy and time to re-resection, adding to the complexity of signature derivation. The novel signature we obtained for response showed components involving genes such as those in the IGF pathway (IGF2BP2, IGF2BP3) and PDGF-signalling pathway (MYC, FLI1, ARHGAP4, JAK3) predictive of poor response to therapy. Likewise, predictors of positive response to therapy included genes involved in the apoptosis and RAS pathways (RAB4A, CHUK) and DNA replication pathways (SSBP2). In sum, this is among the largest cohorts of well-characterized clinical tumor samples for which there is transcriptomic information from primary and re-resected samples from matched patients. Our results not only highlight an innovative computational method for gene signature derivation in the setting of significant underlying heterogeneity, but also result in a predictive gene signature, offering the potential to give therapy to those who stand to benefit most.

Glioblastoma, a very aggressive type of astrocytoma (WHO grade IV), is extremely rare in the pineal region particularly in the pediatric population. With only 5 pediatric cases reported since 1972, we present another report in the setting of technological advances in neurosurgical intervention. As in this case, patients usually present with non-specific symptoms such as headache, nausea, visual changes, and altered mental status. A complete evaluation includes an MRI of the entire neuro-axis. Neurosurgical management of more acute concerns related to increased intracranial pressure can initially consist of a less invasive endoscopic third ventriculostomy (ETV), even if a more invasive microsurgical resection is ultimately required. Furthermore, thorough molecular and histological analyses can guide postoperative adjuvant therapies. Unfortunately, the overall prognosis of a patient with pineal glioblastoma is poor. Despite aggressive treatment with surgery and adjuvant therapies, the average survival rate is less than one year after diagnosis. The potential benefits and risks of a lengthy and complex microsurgical resection of a pineal glioblastoma must be extensively discussed with patients and/or their families. Moreover, due to the aggressive nature of glioblastomas, these patients must be closely monitored postoperatively for progression or recurrence. Though extremely rare, pineal glioblastomas are amenable to neurosurgical intervention for CSF diversion, both ETV and shunt, as well as microsurgical resection. Furthermore, as with any pediatric case, involvement of the patient’s family, pediatrician, and oncologist is crucial in providing comprehensive and long-term supportive care.

Intraocular leiomyomas are uncommon and usually occur in the ciliary body. Primary leiomyoma of the iris is both rare and a difficult diagnosis to make, given melanocytic tumors are more common and may be amelanotic. The somewhat controversial diagnosis of iris leiomyoma requires further confirmation by immunohistochemistry and electron microscopy. Herein, we describe a 58-year-old man with a 2-mm round translucent pink lesion of the iris. The tumor was excised by sector iridectomy. Immunohistochemistry showed positivity for both smooth muscle actin and desmin and negativity for S-100, HMB45, SOX10, MelanA, CD31, CD34, and h-caldesmon. Epstein-Barr virus-associated smooth muscle tumor was excluded by chromogenic in situ hybridization-Epstein-Barr virus-encoded RNA. Ultrastructural analysis showed cytoplasmic myofilaments with focal fusiform densities and micropinocytotic vesicles. Our review of previous literature confirmed the unusual nature of this tumor. Primary iris leiomyoma should be considered in the differential of an amelanotic S-100-immunonegative iris tumor.

Olfactory schwannomas are rare tumors of the anterior skull base that are possibly derived from ectopic Schwann cells, perivascular neural tissue, or sensory nerves of the meninges. The authors report the case of a 14-year-old boy with an olfactory schwannoma that extended inferiorly through the cranial base and superiorly into the frontal lobe. Because of the growth characteristics of the tumor and the significant overlying frontal lobe edema, the lesion was approached via an endonasal endoscopic route, as a strategy to minimize brain retraction. This tumor was characterized radiographically as contrast-enhancing with cystic areas and erosion into bone. The tumor showed immunoreactivity for S100 protein and leukocyte antigen 7 (CD57) but not epithelial membrane antigen, supporting the diagnosis of olfactory schwannoma. A gross-total resection was achieved. This approach represents a novel application of endoscopic endonasal surgery to the pediatric neurosurgical context, as well as a favorable outcome in an extremely unusual tumor type, that should be applicable to other appropriately selected pediatric brain tumors.