PLAG1 fusions define a third subtype of CNS embryonal tumor with PLAG family gene alteration

Abstract

CNS embryonal tumors with PLAGL amplification (ET, PLAGL) are a recently described tumor type marked by amplification of one of the PLAG family genes, PLAGL1 or PLAGL2. Separately, a supratentorial, ependymoma-like CNS tumor type with PLAG family alteration, namely PLAGL1 fusion, was also reported (NET_PLAGL1). Here, we use DNA methylation profiling in combination with copy number, RNA-seq, and histological analysis to characterize and classify a novel group of CNS embryonal tumors harboring PLAG1 gene fusions (n=12). Through our screening, we identified a subset of CNS tumors (n=12) epigenetically distinct from other known CNS tumor types, but clustering close to the PLAGL1- and PLAGL2-amplified ET, PLAGL subtypes in our t-SNE analysis. Copy number profiles indicated putative PLAG1 fusions, which were confirmed in 9/12 tumors (not determined in 3/12). Different 5' fusion partners (ASAP1, ADGRG1, TMEM68, TCF4, CHD7, NCALD, HNRNPK, LOC105378102) were identified that upregulate wild-type PLAG1 through promoter hijacking. Expression analysis shows upregulation of PLAG1 as well as IGF2, DLK1, Desmin, CYP2W1, and RET, which are also robustly expressed in PLAGL1/2-amplified tumors. Patient characteristics, survival data, and clinical/imaging analysis show additional similarities to PLAGL1/2-amplified tumors. Median age at diagnosis was 5 years, tumors were located throughout the neuroaxis, and original histological diagnoses were heterogeneous. The tumors demonstrated morphologic heterogeneity, with most composed of densely cellular areas of primitive small blue cells, alongside focal regions showing clear cell morphology, microcystic changes, and ependymoma-like perivascular pseudorosettes. Applied treatment regimens were also heterogeneous, but some favorable responses to therapy were observed. In summary, we describe a third subtype of PLAG family-altered pediatric CNS embryonal tumor characterized by PLAG1 gene fusion, which leads to upregulation of PLAG1 and downstream genes. We therefore propose to rename ET, PLAGL to ET, PLAG (CNS embryonal tumor with PLAG family gene alteration) together with a specification of the respective subtype.

Keywords: PLAG1; PLAG1 fusion; PLAGL1; PLAGL2; Embryonal CNS tumor.

Fig. 1

Screening of tumors either located in proximity to the ET, PLAGL methylation-based t-SNE cluster or having received a classifier score of ET, PLAGL identifies a novel epigenetically distinct subtype of ET, PLAGL characterized by PLAG1 gene fusions. a Left: DNA methylation-based t-SNE analysis of >140,000 pediatric and adult tumor samples. Marked is the ET, PLAGL type. Right: enlarged depiction of the samples selected for screening and classified as ET, PLAGL, or other tumor types. Methylation classes are color-coded as described in [4]; gray color means that the sample could not be matched to any of the existing methylation classes. b Workflow of the screening process: samples were selected based on t-SNE (proximity to ET, PLAGL) or classifier result (score of ET, PLAGL). Methylation array-derived copy-number data were then screened in the selected samples regarding the PLAG family genes using IGV. c DNA methylation-based analysis using t-SNE dimensionality reduction on 91 PLAG(L)-altered tumors (PLAGL1-amplified, PLAGL2-amplified, PLAG1-fused) and a reference cohort of 3183 different CNS tumors including 1669 gliomas/glioneuronal tumors (HGG, adult and pediatric), 1096 sarcomas, 130 medulloblastomas, 39 supratentorial neuroepithelial tumors with PLAGL1 fusion, and 23 neuroepithelial tumors with PATZ1 fusion. Methylation classes are color-coded and labeled using the respective group abbreviations. ET, PLAGL tumors are differentially colored according to their altered PLAG-family gene. Samples belonging to the ET, PLAGL type are depicted enlarged on the right. Group names are: high-grade glioma (HGG), pleomorphic xanthoastrocytoma (PXA), non-tumor control samples (CTRL), sarcoma, medulloblastoma (MB), high-grade glioma MYCN subtype (HGG-pedMYCN), supratentorial neuroepithelial tumor with PLAGL1 fusion (NET_PLAGL1), neuroepithelial tumor with PATZ1 fusion (PATZ), and low score (samples with a score ≤0.85 for any class)

Fig. 2

Copy-number analysis of CNS embryonal tumors with suspected PLAG1 gene fusion. a Copy-number breakpoints occurring at the PLAG1 locus on chromosome 8q12.1 indicating possible fusion events were visualized using IGV. PLAG1 gene fusions were verified using RNA-seq, WGS, or prior institutional sequencing. Heidelberg classifier methylation class and calibrated score (v12.8) are indicated. b Shown are examples of the fusion caller results, including breakpoints and fusion partners

Fig. 3

Gene expression of CNS embryonal tumors with PLAGL1/2 amplification or PLAG1 fusion. Boxplots comparing gene expression between CNS tumor types for a select set of genes. plag = PLAGL1/2-amplified and PLAG1-fused tumors; pa = pilocytic astrocytoma; pxa = pleomorphic xanthoastrocytoma; hgg = high-grade gliomas (G34R/V, K27M, pedRTK1/2); norm = normal brain tissues; atrt = atypical teratoid rhabdoid tumor; etmr = embryonal tumor with multilayered rosettes; med = medulloblastomas (WNT, SHH, group 3, group 4); red: samples with PLAGL1 amplification, turquois: samples with PLAGL2 amplification, and blue: samples with PLAG1 fusion. Significance bars indicate groups whose differences in gene expression are statistically significant when compared to the first group (plag) (t test, Bonferroni-corrected p value = 0.00714286). PLAG1 upregulation is statistically significant compared to all other groups when looking at PLAG1-fused tumors separately

Fig. 4

Histologic and immunohistochemical features of CNS embryonal tumors with PLAG1 fusion. High-resolution H&E-stained histology images of PLAG1-fused tumors. a, b Focally, the tumors exhibit features reminiscent of embryonal morphology, including densely cellular areas composed of primitive small round blue cells. b A subset of tumors contains follicle-like structures with homogeneous eosinophilic secretions. c, d Other regions demonstrate divergent differentiation, including clear cell changes and microcystic architecture. a, e An ependymoma-like appearance with perivascular pseudorosettes is also noted. f Immunohistochemistry reveals diffuse GFAP positivity. g, h EMA and synaptophysin are negative. i Desmin shows focal cytoplasmic positivity

Fig. 5

Patient characteristics and clinical outcomes of patients with PLAG1-fused CNS embryonal tumors. a Summary of sex, age, localization of tumor, and primary diagnosis. b Kaplan–Meier plots showing progression-free survival (PFS) and overall survival (OS). c Swimmer plot showing a detailed summary of clinical information and outcome for five patients. OS is shown on the x-axis. IDs, age, and primary diagnosis are listed for each patient on the left. Bars are colored according to treatment (CT and RT). Extent of resection, residual tumor amount after resection, and response to treatment were determined through review of MRI scans and displayed through the different symbols. Death of a patient is symbolized by an obelisk behind the respective bar. Abbreviations: emb, embryonal; NOS, not otherwise specified; EPN, ependymoma; LGG, low-grade glioma; HGNET_PLAG1, high-grade neuroepithelial tumor with PLAG1 fusion; Bx, biopsy; Rx, resection; GTR, gross total resection; CT, chemotherapy; HDCT, high-dose chemotherapy; ASCR, autologous stem cell rescue; VP, Vepesid; Carbo, Carboplatin; RT, radiotherapy; CSI, craniospinal irradiation

Fig. 6

Overview of an updated nomenclature of CNS tumors with PLAG family gene alteration including specification of the respective subtype