Multiplatform molecular analyses refine classification of gliomas arising in patients with neurofibromatosis type 1.

Gliomas arising in the setting of neurofibromatosis type 1 (NF1) are heterogeneous, occurring from childhood through adulthood, can be histologically low-grade or high-grade, and follow an indolent or aggressive clinical course. Comprehensive profiling of genetic alterations beyond NF1 inactivation and epigenetic classification of these tumors remain limited. Through next-generation sequencing, copy number analysis, and DNA methylation profiling of gliomas from 47 NF1 patients, we identified 2 molecular subgroups of NF1-associated gliomas. The first harbored biallelic NF1 inactivation only, occurred primarily during childhood, followed a more indolent clinical course, and had a unique epigenetic signature for which we propose the terminology "pilocytic astrocytoma, arising in the setting of NF1". The second subgroup harbored additional oncogenic alterations including CDKN2A homozygous deletion and ATRX mutation, occurred primarily during adulthood, followed a more aggressive clinical course, and was epigenetically diverse, with most tumors aligning with either high-grade astrocytoma with piloid features or various subclasses of IDH-wildtype glioblastoma. Several patients were treated with small molecule MEK inhibitors that resulted in stable disease or tumor regression when used as a single agent, but only in the context of those tumors with NF1 inactivation lacking additional oncogenic alterations. Together, these findings highlight recurrently altered pathways in NF1-associated gliomas and help inform targeted therapeutic strategies for this patient population.

MHC class I loss in endometrial carcinoma: a potential resistance mechanism to immune checkpoint inhibition.

Major histocompatibility complex (MHC) class I is a membrane-bound protein complex expressed on nucleated human cells. MHC class I presents intracellular protein fragments to cytotoxic T cells and triggers an activation cascade upon neoantigen detection by these cells. MHC class I loss by tumor cells decreases tumor neoantigen presentation to the immune system and therefore represents a possible mechanism of immunotherapeutic resistance even among cancers that otherwise appear to be good candidates for checkpoint inhibition, such as mismatch repair (MMR)-deficient and PD-L1-positive malignancies. We herein assess MHC class I expression in a range of endometrial carcinomas, including MMR-deficient and PD-L1-positive cancers. Immunohistochemical staining for combined MHC class I A-, B-, and C-heavy chains was performed on 76 cases of endometrial carcinoma and was classified as present, subclonally lost, or diffusely lost. Tumoral PD-L1 expression, PD-L1 combined positive score, and CD3-positive T lymphocytes were also quantified. Forty-two percent of tumors showed loss of MHC class I expression, either in a subclonal (26%) or diffuse (16%) pattern. This included 46% of MMR-deficient and 25% of PD-L1-positive cancers. These findings suggest that tumoral MHC class I status may be an important factor to consider when selecting endometrial cancer patients for checkpoint inhibition.

The immunohistochemical, DNA methylation, and chromosomal copy number profile of cauda equina paraganglioma is distinct from extra-spinal paraganglioma.

Paragangliomas are neuroendocrine tumors of the autonomic nervous system that are variably clinically functional and have a potential for metastasis. Up to 40% occur in the setting of a hereditary syndrome, most commonly due to germline mutations in succinate dehydrogenase (SDHx) genes. Immunohistochemically, paragangliomas are characteristically GATA3-positive and cytokeratin-negative, with loss of SDHB expression in most hereditary cases. In contrast, the rare paragangliomas arising in the cauda equina (CEP) or filum terminale region have been shown to be hormonally silent, clinically indolent, and have variable keratin expression, suggesting these tumors may represent a separate pathologic entity. We retrospectively evaluated 17 CEPs from 11 male and 6 female patients with a median age of 38 years (range 21-82), none with a family history of neuroendocrine neoplasia. Six of the 17 tumors demonstrated prominent gangliocytic or ganglioneuromatous differentiation. By immunohistochemistry, none of the CEPs showed GATA3 positivity or loss of SDHB staining; all 17 CEPs were cytokeratin positive. Genome-wide DNA methylation profiling was performed on 12 of the tumors and compared with publicly available genome-wide DNA methylation data. Clustering analysis showed that CEPs form a distinct epigenetic group, separate from paragangliomas of extraspinal sites, pheochromocytomas, and other neuroendocrine neoplasms. Copy number analysis revealed diploid genomes in the vast majority of CEPs, whereas extraspinal paragangliomas were mostly aneuploid with recurrent trisomy 1q and monosomies of 1p, 3, and 11, none of which were present in the cohort of CEP. Together, these findings indicate that CEPs likely represent a distinct entity. Future genomic studies are needed to further elucidate the molecular pathogenesis of these tumors.

Mismatch repair status and PD-L1 expression in clear cell carcinomas of the ovary and endometrium.

Clear cell carcinoma represents a distinct histologic type of müllerian carcinoma that is resistant to conventional chemotherapy. Expression of programmed cell death ligand (PD-L1) has been associated with immune evasion in numerous tumor types and may be used to identify patients who will benefit from targeted immunotherapy, particularly in the setting of mismatch repair defects. We evaluated PD-L1 expression in 23 ovarian clear cell carcinomas and 21 endometrial clear cell carcinomas, and correlated expression with mismatch repair status. Tumor PD-L1 staining was seen in 43% of ovarian tumors and 76% of endometrial tumors, including 71% of cases (67% of ovarian and 75% of endometrial) with mismatch repair defects. Extensive tumoral staining (>50%) was seen in only one case (an endometrial case with MSH6 loss). However, tumoral PD-L1 expression remained common in mismatch repair-intact tumors and mismatch repair status was not significantly correlated with PD-L1 expression. The increased incidence of PD-L1 positivity in tumor cells (P=0.04) in endometrial vs ovarian clear cell carcinomas suggests differences in the tumor microenvironment of these histologically and molecularly similar tumors that may inform treatment options. These results suggest that clear cell histology may be a useful susceptibility marker for immunotherapy targeting the PD-1/PD-L1 axis irrespective of mismatch repair status, particularly in endometrial carcinomas.

Mucinous Differentiation With Tumor Infiltrating Lymphocytes Is a Feature of Sporadically Methylated Endometrial Carcinomas.

Defects in the DNA mismatch-repair system are identified in ∼25%-30% of endometrial carcinomas (ECs). Whereas some ECs are due to germline Lynch syndrome (LS)-associated mutations, the majority demonstrate sporadic MLH1 promoter hypermethylation (MLH1hm). MLH1hm characterizes a unique subset of colorectal cancers with a relatively poor prognosis; however, the morphology and behavior of sporadically methylated ECs (SMECs) are less well understood. We herein review the clinicopathologic features of 34 SMECs diagnosed at The University of Virginia and compare them with LS-associated and Lynch-like endometrial cancers. Most SMECs were centered in the uterine fundus (74%) and displayed Grade 1 (35%) or Grade 2 (41%) endometrioid morphology. Mucinous differentiation was present in 50%, and 65% showed tumor-infiltrating lymphocytes. Cases with undifferentiated components or sarcomatous differentiation were relatively rare (9%). Most SMECs presented at stage 1 or 2 (91%). When compared with LS and Lynch-like endometrial cancers, SMECs occurred ∼8 yr later and were significantly more likely to show conventional endometrioid morphology (P=0.04), mucinous differentiation (P=0.002), and tumor-infiltrating lymphocytes (P=0.002). These findings suggest that SMECs constitute a clinicopathologically distinct subset of mismatch-repair-deficient/high-level microsatellite instability tumors. Future studies are needed to further understand the implications of MLH1hm on the prognosis and treatment of endometrial cancer.

Limited nucleotide changes in the Rev response element (RRE) during HIV-1 infection alter overall Rev-RRE activity and Rev multimerization.

HIV-1 Rev and the Rev response element (RRE) enable a critical step in the viral replication cycle by facilitating the nuclear export of intron-containing mRNAs, yet their activities have rarely been analyzed in natural infections. This study characterized their genetic and functional variation in a small cohort of HIV-infected individuals. Multiple Rev and RRE sequences were obtained using single-genome sequencing (SGS) of plasma samples collected within 6 months after seroconversion and at a later time. This allowed the identification of cognate sequences that were linked in vivo in the same viral genome and acted together as a functional unit. Phylogenetic analyses of these sequences indicated that 4/5 infections were founded by a single transmission event. Rev and RRE variants from each time point were subjected to functional analysis as both cognate pairs and as individual components. While a range of Rev-RRE activities were seen, the activity of cognate pairs from a single time point clustered to a discrete level, which was termed the set point. In 3/5 patients, this set point changed significantly over the time period studied. In all patients, RRE activity was more sensitive to sequence variation than Rev activity and acted as the primary driver of the cognate set point. Selected patient RREs were also shown to have differences in Rev multimerization using gel shift binding assays. Thus, rather than acting as a simple on-off switch or maintaining a constant level of activity throughout infection, the Rev-RRE system can fluctuate, presumably to control replication.

Single-nucleotide changes in the HIV Rev-response element mediate resistance to compounds that inhibit Rev function.

Previously we described the identification of two compounds (3-amino-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide [103833] and 4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile [104366]) that interfered with HIV replication through the inhibition of Rev function. We now describe resistant viral variants that arose after drug selection, using virus derived from two different HIV proviral clones, NL4-3 and R7/3. With HIV(NL4-3), each compound selected a different single point mutation in the Rev response element (RRE) at the bottom of stem-loop IIC. Either mutation led to the lengthening of the stem-loop IIC stem by an additional base pair, creating an RRE that was more responsive to lower concentrations of Rev than the wild type. Surprisingly, wild-type HIV(R7/3) was also found to be inhibited when tested with these compounds, in spite of the fact this virus already has an RNA stem-loop IIC similar to the one in the resistant NL4-3 variant. When drug resistance was selected in HIV(R7/3), a virus arose with two nucleotide changes that mapped to the envelope region outside the RRE. One of these nucleotide changes was synonymous with respect to env, and one was not. The combination of both nucleotide changes appeared to be necessary for the resistance phenotype as the individual point mutations by themselves did not convey resistance. Thus, although drug-resistant variants can be generated with both viral strains, the underlying mechanism is clearly different. These results highlight that minor nucleotide changes in HIV RNA, outside the primary Rev binding site, can significantly alter the efficiency of the Rev/RRE pathway.

A Case of Neurosarcoid Presenting as Multiple Intraparenchymal Hemorrhages.

This report explores the case of a 49-year-old African American male with a six-month history of multifocal neurological deficits who presented to an outside hospital after a generalized seizure. Patient was transferred to our tertiary medical center after brain imaging showed multiple bilateral supratentorial intraparenchymal hemorrhages (IPH). A brain biopsy confirmed parenchymal and perivascular non-caseating granulomas with vasculitis. The patient was definitively diagnosed with neurosarcoidosis (NS) and his condition improved with high dose corticosteroids and additional immunosuppressive therapies. Intracranial hemorrhage in the setting of NS is extremely rare, with fewer than thirty documented cases; however, this is likely an underestimation of its true prevalence. This case illustrates the difficulty in diagnosis as many other etiologies of IPH must be considered. Additionally, the clinical course and manifestations of NS is often quite variable. The uniqueness of this case lies in the rapid progression from seemingly incidental microhemorrhages to multiple large IPHs over two months. While the cause of this progression is not immediately apparent, a possible cause may be inadequate initial treatment due to delayed diagnosis. Our case demonstrates the importance of early recognition and initiation of immunosuppressive therapy, potentially leading to dramatic clinical improvement, as seen in this patient.

Remarkable Intracranial Response to Sotorasib in a Patient With KRAS G12C-Mutated Lung Adenocarcinoma and Untreated Brain Metastases: A Case Report.

Sotorasib is a KRAS G12C inhibitor that recently received approval for use in locally advanced or metastatic KRAS G12C-mutated NSCLC. CodeBreaK100, the phase 2 clinical trial leading to the approval of sotorasib, excluded patients with untreated brain metastases; there have been no reports describing efficacy of sotorasib on untreated brain metastases. We present a case of a patient with active untreated brain metastases with resulting disorientation and weakness who has radiographic response and complete resolution of neurologic symptoms with sotorasib. Our case illustrates the intracranial activity of sotorasib, but additional studies are needed to characterize the intracranial response rate and duration of response in these patients.

Intracranial mesenchymal tumors with FET-CREB fusion are composed of at least two epigenetic subgroups distinct from meningioma and extracranial sarcomas.

'Intracranial mesenchymal tumor, FET-CREB fusion-positive' occurs primarily in children and young adults and has previously been termed intracranial angiomatoid fibrous histiocytoma (AFH) or intracranial myxoid mesenchymal tumor (IMMT). Here we performed genome-wide DNA methylation array profiling of 20 primary intracranial mesenchymal tumors with FET-CREB fusion to further study their ontology. These tumors resolved into two distinct epigenetic subgroups that were both divergent from all other analyzed intracranial neoplasms and soft tissue sarcomas, including meningioma, clear cell sarcoma of soft tissue (CCS), and AFH of extracranial soft tissue. The first subgroup (Group A, 16 tumors) clustered nearest to but independent of solitary fibrous tumor and AFH of extracranial soft tissue, whereas the second epigenetic subgroup (Group B, 4 tumors) clustered nearest to but independent of CCS and also lacked expression of melanocytic markers (HMB45, Melan A, or MITF) characteristic of CCS. Group A tumors most often occurred in adolescence or early adulthood, arose throughout the neuroaxis, and contained mostly EWSR1-ATF1 and EWSR1-CREB1 fusions. Group B tumors arose most often in early childhood, were located along the cerebral convexities or spinal cord, and demonstrated an enrichment for tumors with CREM as the fusion partner (either EWSR1-CREM or FUS-CREM). Group A tumors more often demonstrated stellate/spindle cell morphology and hemangioma-like vasculature, whereas Group B tumors more often demonstrated round cell or epithelioid/rhabdoid morphology without hemangioma-like vasculature, although robust comparison of these clinical and histologic features requires future study. Patients with Group B tumors had inferior progression-free survival relative to Group A tumors (median 4.5 vs. 49 months, p = 0.001). Together, these findings confirm that intracranial AFH-like neoplasms and IMMT represent histologic variants of a single tumor type ('intracranial mesenchymal tumor, FET-CREB fusion-positive') that is distinct from meningioma and extracranial sarcomas. Additionally, epigenomic evaluation may provide important prognostic subtyping for this unique tumor entity.

Intracranial temporal bone angiomatoid fibrous histiocytoma: illustrative case.

BACKGROUND: Angiomatoid fibrous histiocytoma (AFH) is a rare, slowly progressive neoplasm that most commonly occurs in soft tissues. AFH rarely occurs in bone such as the calvaria. The authors present a case of AFH in the petrous temporal bone, which, to their knowledge, is the first case of AFH in this location. OBSERVATIONS: A 17-year-old girl presented with worsening positional headaches with associated tinnitus and hearing loss. Imaging demonstrated an extraaxial mass extending into the right cerebellopontine angle, with erosion of the petrous temporal bone, with features atypical for a benign process. The patient underwent retrosigmoid craniotomy for tumor resection. Pathology was consistent with a spindle cell tumor, and genetic testing further revealed an EWSR1 gene rearrangement, confirming the diagnosis of AFH. The patient was discharged with no complications. Her symptoms have resolved, and surveillance imaging has shown no evidence of recurrence. LESSONS: The authors report the first case of AFH in the petrous temporal bone and only the second known case in the calvaria. This case illustrates the importance of the resection of masses with clinical and imaging features atypical of more benign entities such as meningiomas. It is important to keep AFH in the differential diagnosis for atypical masses in the calvaria and skull base.

Detection of glioma infiltration at the tumor margin using quantitative stimulated Raman scattering histology.

In the management of diffuse gliomas, the identification and removal of tumor at the infiltrative margin remains a central challenge. Prior work has demonstrated that fluorescence labeling tools and radiographic imaging are useful surgical adjuvants with macroscopic resolution. However, they lose sensitivity at the tumor margin and have limited clinical utility for lower grade histologies. Fiber-laser based stimulated Raman histology (SRH) is an optical imaging technique that provides microscopic tissue characterization of unprocessed tissues. It remains unknown whether SRH of tissues taken from the infiltrative glioma margin will identify microscopic residual disease. Here we acquired glioma margin specimens for SRH, histology, and tumor specific tissue characterization. Generalized linear mixed models were used to evaluate agreement. We find that SRH identified residual tumor in 82 of 167 margin specimens (49%), compared to IHC confirming residual tumor in 72 of 128 samples (56%), and H&E confirming residual tumor in 82 of 169 samples (49%). Intraobserver agreements between all 3 modalities were confirmed. These data demonstrate that SRH detects residual microscopic tumor at the infiltrative glioma margin and may be a promising tool to enhance extent of resection.

Intracranial mesenchymal tumor with FET-CREB fusion-A unifying diagnosis for the spectrum of intracranial myxoid mesenchymal tumors and angiomatoid fibrous histiocytoma-like neoplasms.

Intracranial mesenchymal tumors with FET-CREB fusions are a recently described group of neoplasms in children and young adults characterized by fusion of a FET family gene (usually EWSR1, but rarely FUS) to a CREB family transcription factor (ATF1, CREB1, or CREM), and have been variously termed intracranial angiomatoid fibrous histiocytoma or intracranial myxoid mesenchymal tumor. The clinical outcomes, histologic features, and genomic landscape are not well defined. Here, we studied 20 patients with intracranial mesenchymal tumors proven to harbor FET-CREB fusion by next-generation sequencing (NGS). The 16 female and four male patients had a median age of 14 years (range 4-70). Tumors were uniformly extra-axial or intraventricular and located at the cerebral convexities (n = 7), falx (2), lateral ventricles (4), tentorium (2), cerebellopontine angle (4), and spinal cord (1). NGS demonstrated that eight tumors harbored EWSR1-ATF1 fusion, seven had EWSR1-CREB1, four had EWSR1-CREM, and one had FUS-CREM. Tumors were uniformly well circumscribed and typically contrast enhancing with solid and cystic growth. Tumors with EWSR1-CREB1 fusions more often featured stellate/spindle cell morphology, mucin-rich stroma, and hemangioma-like vasculature compared to tumors with EWSR1-ATF1 fusions that most often featured sheets of epithelioid cells with mucin-poor collagenous stroma. These tumors demonstrated polyphenotypic immunoprofiles with frequent positivity for desmin, EMA, CD99, MUC4, and synaptophysin, but absence of SSTR2A, myogenin, and HMB45 expression. There was a propensity for local recurrence with a median progression-free survival of 12 months and a median overall survival of greater than 60 months, with three patients succumbing to disease (all with EWSR1-ATF1 fusions). In combination with prior case series, this study provides further insight into intracranial mesenchymal tumors with FET-CREB fusion, which represent a distinct group of CNS tumors encompassing both intracranial myxoid mesenchymal tumor and angiomatoid fibrous histiocytoma-like neoplasms.

Comprehensive analysis of diverse low-grade neuroepithelial tumors with FGFR1 alterations reveals a distinct molecular signature of rosette-forming glioneuronal tumor.

The FGFR1 gene encoding fibroblast growth factor receptor 1 has emerged as a frequently altered oncogene in the pathogenesis of multiple low-grade neuroepithelial tumor (LGNET) subtypes including pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor (DNT), rosette-forming glioneuronal tumor (RGNT), and extraventricular neurocytoma (EVN). These activating FGFR1 alterations in LGNET can include tandem duplication of the exons encoding the intracellular tyrosine kinase domain, in-frame gene fusions most often with TACC1 as the partner, or hotspot missense mutations within the tyrosine kinase domain (either at p.N546 or p.K656). However, the specificity of these different FGFR1 events for the various LGNET subtypes and accompanying genetic alterations are not well defined. Here we performed comprehensive genomic and epigenomic characterization on a diverse cohort of 30 LGNET with FGFR1 alterations. We identified that RGNT harbors a distinct epigenetic signature compared to other LGNET with FGFR1 alterations, and is uniquely characterized by FGFR1 kinase domain hotspot missense mutations in combination with either PIK3CA or PIK3R1 mutation, often with accompanying NF1 or PTPN11 mutation. In contrast, EVN harbors its own distinct epigenetic signature and is characterized by FGFR1-TACC1 fusion as the solitary pathogenic alteration. Additionally, DNT and pilocytic astrocytoma are characterized by either kinase domain tandem duplication or hotspot missense mutations, occasionally with accompanying NF1 or PTPN11 mutation, but lacking the accompanying PIK3CA or PIK3R1 mutation that characterizes RGNT. The glial component of LGNET with FGFR1 alterations typically has a predominantly oligodendroglial morphology, and many of the pilocytic astrocytomas with FGFR1 alterations lack the biphasic pattern, piloid processes, and Rosenthal fibers that characterize pilocytic astrocytomas with BRAF mutation or fusion. Together, this analysis improves the classification and histopathologic stratification of LGNET with FGFR1 alterations.

Neurologic phenotype of Schimke immuno-osseous dysplasia and neurodevelopmental expression of SMARCAL1.

Schimke immuno-osseous dysplasia (OMIM 242900) is an uncommon autosomal-recessive multisystem disease caused by mutations in SMARCAL1 (swi/snf-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), a gene encoding a putative chromatin remodeling protein. Neurologic manifestations identified to date relate to enhanced atherosclerosis and cerebrovascular disease. Based on a clinical survey, we determined that half of Schimke immuno-osseous dysplasia patients have a small head circumference, and 15% have social, language, motor, or cognitive abnormalities. Postmortem examination of 2 Schimke immuno-osseous dysplasia patients showed low brain weights and subtle brain histologic abnormalities suggestive of perturbed neuron-glial migration such as heterotopia, irregular cortical thickness, incomplete gyral formation, and poor definition of cortical layers. We found that SMARCAL1 is highly expressed in the developing and adult mouse and human brain, including neural precursors and neuronal lineage cells. These observations suggest that SMARCAL1 deficiency may influence brain development and function in addition to its previously recognized effect on cerebral circulation.

Chromatin remodeling and human disease.

In the past few years, there has been a nascent convergence of scientific understanding of inherited human diseases with epigenetics. Identified epigenetic processes involved in human disease include covalent DNA modifications, covalent histone modifications, and histone relocation. Each of these processes influences chromatin structure and thereby regulates gene expression and DNA methylation, replication, recombination, and repair. The importance of these processes for nearly all aspects of normal growth and development is illustrated by the array of multi-system disorders and neoplasias caused by their dysregulation.