Faculty Bibliography

The Publisher regrets that this article is an accidental duplication of an article that has already been published, https://doi.org/10.1016/j.ajpath.2020.07.001. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Glioblastoma, an incurable tumor, remains difficult to model and more importantly to treat due to its genetic/epigenetic heterogeneity and plasticity across cellular states. The ability of current tumor models to recapitulate the cellular states found in primary tumors remains unexplored. To address this issue, we compared single-cell RNA-sequencing of tumor cells from five patients across four patient-specific glioblastoma stem cell (GSC)-derived model types, including glioma spheres, tumor organoids, glioblastoma cerebral organoids (GLICO), and patient-derived xenografts. We find that GSCs within the GLICO model are enriched for a neural progenitor-like cell (NPC) subpopulation and recapitulate the cellular states and their plasticity found in the corresponding primary parental tumors. These data demonstrate how the contribution of a neuroanatomically accurate human microenvironment is critical and sufficient for recapitulating the cellular states found in human primary GBMs, a principle that may likely apply to other tumor models.

Of the four primary subgroups of medulloblastoma, the most frequent cytogenetic abnormality, i17q, distinguishes Groups 3 and 4 which carry the highest mortality; haploinsufficiency of 17p13.3 is a marker for particularly poor prognosis. At the terminal end of this locus lies miR-1253, a brain-enriched microRNA that regulates bone morphogenic proteins during cerebellar development. We hypothesized miR-1253 confers novel tumor-suppressive properties in medulloblastoma. Using two different cohorts of medulloblastoma samples, we first studied the expression and methylation profiles of miR-1253. We then explored the anti-tumorigenic properties of miR-1253 in parallel with a biochemical analysis of apoptosis and proliferation, and isolation of oncogenic targets using high-throughput screening. Deregulation of miR-1253 expression was noted, both in medulloblastoma clinical samples and cell lines, by epigenetic silencing via hypermethylation; specific de-methylation of miR-1253 not only resulted in rapid recovery of expression but also a sharp decline in tumor cell proliferation and target gene expression. Expression restoration also led to a reduction in tumor cell virulence, concomitant with activation of apoptotic pathways, cell cycle arrest, and reduction of markers of proliferation. We identified two oncogenic targets of miR-1253, CDK6 and CD276, whose silencing replicated the negative trophic effects of miR-1253. These data reveal novel tumor-suppressive properties for miR-1253, i.e. 1) loss of expression via epigenetic silencing, 2) negative trophic effects on tumor aggressiveness, and 3) downregulation of oncogenic targets.

Infant high grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histological review, methylation profiling, custom panel and genome/exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an 'intrinsic' spectrum of disease specific to the infant population. These included those with targetable MAP-kinase alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n=31), NTRK1/2/3 (n=21), ROS1 (n=9) and MET (n=4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly supports the concept that infant gliomas require a change in diagnostic practice and management.

Chordoid meningioma is a rare histological variant, which comprises <1% of all meningiomas. In the initially reported case series, these tumors were described to have a high recurrence rate, especially following subtotal resection, and are thus designated as WHO grade 2, according to current diagnostic criteria. However, more recent case series suggested that chordoid morphology itself, in the absence of other atypical features, did not predict more adverse outcomes, and survival paralleled that of all meningiomas. Among a cohort of 1897 meningiomas resected between 1995 and 2019, we identified 11 cases of meningioma with predominant chordoid morphology and sufficient follow up. Nine of those were otherwise WHO grade I, and only one of them recurred. The two remaining cases otherwise qualified for atypical meningioma, one of which recurred and the patient died of disease. Compared to a control cohort of 473 nonchordoid meningiomas with available follow up, chordoid meningiomas that were otherwise WHO grade 1 had progression-free and overall survival of 100%, which paralleled nonchordoid grade 1 meningiomas by Kaplan-Meier survival curve analysis. The follow up interval ranged between 31 and 219 months, with mean follow up of 108 months. Moreover, genome wide DNA methylation profiling of these 9 chordoid cases classified 8 into methylation class Ben-2 category and only 1 into methylation class Int-A category. In summary, our own institutional experience suggests that chordoid histology alone does not portend a worse prognosis. Based on the methylation and survival data, we suggest that chordoid meningiomas should be graded analogously to nonchordoid meningiomas

The Genomics of Malignant Peripheral Nerve Sheath Tumor (GeM) Consortium is an international collaboration focusing on multi-omic analysis of malignant peripheral nerve sheath tumors (MPNSTs), the most aggressive tumor associated with neurofibromatosis type 1 (NF1). Here we present a summary of current knowledge gaps, a description of our consortium and the cohort we have assembled, and an overview of our plans for multi-omic analysis of these tumors. We propose that our analysis will lead to a better understanding of the order and timing of genetic events related to MPNST initiation and progression. Our ten institutions have assembled 96 fresh frozen NF1-related (63%) and sporadic MPNST specimens from 86 subjects with corresponding clinical and pathological data. Clinical data have been collected as part of the International MPNST Registry. We will characterize these tumors with bulk whole genome sequencing, RNAseq, and DNA methylation profiling. In addition, we will perform multiregional analysis and temporal sampling, with the same methodologies, on a subset of nine subjects with NF1-related MPNSTs to assess tumor heterogeneity and cancer evolution. Subsequent multi-omic analyses of additional archival specimens will include deep exome sequencing (500×) and high density copy number arrays for both validation of results based on fresh frozen tumors, and to assess further tumor heterogeneity and evolution. Digital pathology images are being collected in a cloud-based platform for consensus review. The result of these efforts will be the largest MPNST multi-omic dataset with correlated clinical and pathological information ever assembled.

Sudden unexplained death in childhood (SUDC) affects children >1-year-old whose cause of death remains unexplained following comprehensive case investigation and is often associated with hippocampal abnormalities. We prospectively performed systematic neuropathologic investigation in 20 SUDC cases, including (i) autopsy data and comprehensive ancillary testing, including molecular studies, (ii) ex vivo 3T MRI and extensive histologic brain samples, and (iii) blinded neuropathology review by 2 board-certified neuropathologists. There were 12 girls and 8 boys; median age at death was 33.3 months. Twelve had a history of febrile seizures, 85% died during apparent sleep and 80% in prone position. Molecular testing possibly explained 3 deaths and identified genetic mutations in TNNI3, RYR2, and multiple chromosomal aberrations. Hippocampal abnormalities most often affected the dentate gyrus (altered thickness, irregular configuration, and focal lack of granule cells), and had highest concordance between reviewers. Findings were identified with similar frequencies in cases with and without molecular findings. Number of seizures did not correlate with hippocampal findings. Hippocampal alterations were the most common finding on histological review but were also found in possibly explained deaths. The significance and specificity of hippocampal findings is unclear as they may result from seizures, contribute to seizure pathogenesis, or be an unrelated phenomenon.

Background: Clinicopathologic criteria exist to identify synchronous and metastatic endometrioid carcinomas involving endometrium and ovary. Recent studies utilizing next generation sequencing demonstrated that most clinically suspected synchronous ovarian and endometrial endometrioid tumors are in fact clonally related. We sought to define epigenetic signatures of FIGO grade 1 endometrioid carcinomas of endometrial primary, ovarian primary, synchronous endometrial and ovarian primaries, and endometrial primary with ovarian metastasis.
Design(s): DNA was extracted from microdissected formalin-fixed, paraffin-embedded tumor tissues from 8 isolated endometrial primaries, 6 isolated ovarian primaries, 5 synchronous endometrial and ovarian primaries and 3 endometrial primaries with ovarian metastasis and subjected to methylation profiling (Illumina MethylationEPIC array). Methylation data were analyzed with the R Bioconductor package minfi, including quality control, data normalization and differentially methylated CpG site analysis. Subsequent filtering was performed using a p-value cutoff = 0.01 and a minimal mean difference of the Beta-value of = 0.1. Copy number alterations were analyzed using conumee package.
Result(s): Epigenetic profiling revealed that isolated primary endometrial and ovarian tumors formed two distinct methylation clusters according to their site of origin (Fig. 1). Similarly, 4/5 synchronous endometrial and ovarian tumors primary pairs clustered away from each other and by primary site. Both endometrial and ovarian tumors in the remaining synchronous primary pair clustered with isolated primary ovarian tumors. Finally, endometrial and ovarian tumors in all 3 endometrial primaries with ovarian metastasis clustered by disease site. (Fig. 1). Copy number changes largely recapitulated the methylation patterns with some synchronous tumors showing similar profiles and some showing large differences (Fig. 2). Certain copy number alterations (most notably 1q gain) seemed specific to ovarian tumors, a finding observed across both isolated primary and synchronous tumors. (Figure presented)
Conclusion(s): DNA methylation profiles of synchronous endometrial and ovarian tumors and endometrial primaries with ovarian metastasis are similar and cluster by disease site. Copy number changes recapitulate methylation results. These findings suggest site specific effects on tumor development

Background: Molecular screening for therapeutically targetable alterations is considered standard of care in the management of non-small cell lung cancer. However, most molecular assays utilize tumor tissue, which may not always be available. This has led to the development of "liquid biopsies": Plasma-based Next Generation Sequencing (NGS) tests that use circulating tumor DNA as a substrate to identify relevant targets. In this study, we sought to determine the level of agreement between the two tests as they are used in clinical practice and to investigate the utility of concurrent plasma/tissue testing.
Design(s): We identified 47 cases of lung adenocarcinoma diagnosed over the past 2 years, who received concurrent testing (within 24 weeks) with both our institution's tissue (DNA and RNA based) NGS assay and a commercial plasma-based NGS assay. The results were reviewed to establish concordance in the identification of mutations or fusions deemed clinically relevant or for which a targeted therapy was available.
Result(s): Patients in our cohort represented both new diagnoses (31 cases, 66%) and disease progression on treatment (16 cases, 34%). The majority (83%) had stage 4 disease. Tissue NGS identified clinically relevant mutations in 39 cases (83%), including in 14 (88%) of the previously treated cases. By comparison, plasma NGS identified clinically relevant mutations in 20 cases (43%, p<0.001), including 6 treated cases (38%, p=0.01). Tissue NGS identified therapeutic targets in 55% of cases and 75% of previously treated cases; while plasma NGS identified targets in 28% and 25% respectively (p<0.001 and p=0.01 respectively). All clinically relevant mutations identified by plasma NGS were also detected by tissue NGS, while plasma NGS detected only 51% those identified by tissue NGS. Discrepant cases involved hotspot mutations and actionable fusions including those in EGFR, KRAS, and ROS1 (Table 1).(Table presented)
Conclusion(s): Tissue NGS detects more clinically relevant alterations and therapeutic targets compared to plasma NGS, especially in the post-treatment setting, suggesting that tissue NGS should be the preferred method for molecular testing of lung adenocarcinoma. Additionally, all clinically relevant mutations identified by plasma NGS were also detected by tissue NGS, suggesting that tissue/plasma cotesting provides little additional benefit over tissue NGS alone. Plasma NGS can detect clinically relevant targets, and still plays an important role when tissue testing is impractical or not possible