Faculty Bibliography

BACKGROUND:Only few data are available on treatment-associated behavior of distinct rare CNS-embryonal tumor entities previously treated as "CNS-primitive neuroectodermal tumors" (CNS-PNET). Respective data on specific entities, including CNS neuroblastoma, FOXR2 activated (CNS NB-FOXR2), and embryonal tumor with multi-layered rosettes (ETMR) are needed for development of differentiated treatment strategies. METHODS:Within this retrospective, international study, tumor samples of clinically well-annotated patients with the original diagnosis of CNS-PNET were analyzed using DNA methylation arrays (n=307). Additional cases (n=66) with DNA methylation pattern of CNS NB-FOXR2 were included irrespective of initial histological diagnosis. Pooled clinical data (n=292) were descriptively analyzed. RESULTS:DNA methylation profiling of "CNS-PNET" classified 58(19%) cases as ETMR, 57(19%) as HGG, 36(12%) as CNS NB-FOXR2, and 89(29%) cases were classified into 18 other entities. Sixty-seven (22%) cases did not show DNA methylation patterns similar to established CNS tumor reference classes. Best treatment results were achieved for CNS NB-FOXR2 patients (5-year PFS: 63%±7%, OS: 85%±5%, n=63), with 35/42 progression-free survivors after upfront craniospinal irradiation (CSI) and chemotherapy. The worst outcome was seen for ETMR and HGG patients with 5-year PFS of 18%±6% and 22%±7%, and 5-year OS of 24%±6% and 25%±7%, respectively. CONCLUSION/CONCLUSIONS:The historically reported poor outcome of CNS-PNET patients becomes highly variable when tumors are molecularly classified based on DNA methylation profiling. Patients with CNS NB-FOXR2 responded well to current treatments and a standard-risk-CSI based regimen may be prospectively evaluated. The poor outcome of ETMR across applied treatment strategies substantiates the necessity for evaluation of novel treatments.

Molecular groups of supratentorial ependymomas comprise tumors with ZFTA-RELA or YAP1-involving fusions and fusion-negative subependymoma. However, occasionally supratentorial ependymomas cannot be readily assigned to any of these groups due to lack of detection of a typical fusion and/or ambiguous DNA methylation-based classification. An unbiased approach with a cohort of unprecedented size revealed distinct methylation clusters composed of tumors with ependymal but also various other histological features containing alternative translocations that shared ZFTA as a partner gene. Somatic overexpression of ZFTA-associated fusion genes in the developing cerebral cortex is capable of inducing tumor formation in vivo, and cross-species comparative analyses identified GLI2 as a key downstream regulator of tumorigenesis in all tumors. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating a potential therapeutic vulnerability in ZFTA fusion-positive tumors.

Glioblastoma IDH-wildtype presents with a wide histological spectrum. Some features are so distinctive that they are considered as separate histological variants or patterns for the purpose of classification. However, these usually lack defined (epi-)genetic alterations or profiles correlating with this histology. Here, we describe a molecular subtype with overlap to the unique histological pattern of glioblastoma with primitive neuronal component. Our cohort consists of 63 IDH-wildtype glioblastomas that harbor a characteristic DNA methylation profile. Median age at diagnosis was 59.5 years. Copy-number variations and genetic sequencing revealed frequent alterations in TP53, RB1 and PTEN, with fewer gains of chromosome 7 and homozygous CDKN2A/B deletions than usually described for IDH-wildtype glioblastoma. Gains of chromosome 1 were detected in more than half of the cases. A poorly differentiated phenotype with frequent absence of GFAP expression, high proliferation index and strong staining for p53 and TTF1 often caused misleading histological classification as carcinoma metastasis or primitive neuroectodermal tumor. Clinically, many patients presented with leptomeningeal dissemination and spinal metastasis. Outcome was poor with a median overall survival of only 12 months. Overall, we describe a new molecular subtype of IDH-wildtype glioblastoma with a distinct histological appearance and genetic signature.

Retinoblastoma is a childhood cancer of the retina involving germline or somatic alterations of the RB Transcriptional Corepressor 1 gene, RB1. Rare cases of sellar-suprasellar region retinoblastoma without evidence of ocular or pineal tumors have been described. A nine-month-old male presented with a sellar-suprasellar region mass. Histopathology showed an embryonal tumor with focal Flexner-Wintersteiner-like rosettes and loss of retinoblastoma protein (RB1) expression by immunohistochemistry. DNA array-based methylation profiling confidently classified the tumor as pineoblastoma group A/intracranial retinoblastoma. The patient was subsequently enrolled on an institutional translational cancer research protocol and underwent comprehensive molecular profiling, including paired tumor/normal exome and genome sequencing and RNA-sequencing of the tumor. Additionally, Pacific Biosciences (PacBio) Single Molecule Real Time (SMRT) sequencing was performed from comparator normal and disease-involved tissue to resolve complex structural variations. RNA-sequencing revealed multiple fusions clustered within 13q14.1-q21.3, including a novel in-frame fusion of RB1-SIAH3 predicted to prematurely truncate the RB1 protein. SMRT sequencing revealed a complex structural rearrangement spanning 13q14.11-q31.3, including two somatic structural variants within intron 17 of RB1. These events corresponded to the RB1-SIAH3 fusion and a novel RB1 rearrangement expected to correlate with the complete absence of RB1 protein expression. Comprehensive molecular analysis, including DNA array-based methylation profiling and sequencing-based methodologies, were critical for classification and understanding the complex mechanism of RB1 inactivation in this diagnostically challenging tumor.

This is the first report of a NACC2-NTRK2 fusion in a histological glioblastoma. Oncogenomic analysis revealed this actionable fusion oncogene in a pediatric cerebellar glioblastoma, which would not have been identified through routine diagnostics, demonstrating the value of clinical genome profiling in cancer care.

Chromosomal instability due to mutations in genes guarding the stability of the genome is a well-known mechanism underlying tumorigenesis and malignant progression in numerous cancers. The effect of this process in gliomas is mostly unknown with relatively little research examining the effects of chromosomal instability on patient outcome and therapeutic efficacy, although studies have shown that overall/total copy number variation (CNV) is elevated in higher histologic grades and in cases with more rapid progression and shorter patient survival. Herein, we examine a 70-gene mRNA expression signature (CIN70), which has been previously shown to correlate tightly with chromosomal instability, in 2 independent cohorts of IDH-mutant astrocytomas (total n = 241), IDH-wildtype astrocytomas (n = 228), and oligodendrogliomas (n = 128). Our results show that CIN70 expression levels correlate with total CNV, as well as higher grade, progression-free survival, and overall survival in both IDH-mutant and IDH-wildtype astrocytomas. In oligodendrogliomas, these mRNA signatures correlate with total CNV but not consistently with clinical outcome. These data suggest that chromosomal instability is an underlying factor in aggressive behavior and progression of a subset of diffuse astrocytomas. In addition, chromosomal instability may in part explain the poor response of diffuse gliomas to treatment and may serve as a future therapeutic target.

Encapsulated papillary carcinomas (EPC) of the breast is a unique variant of papillary carcinoma confined to a cystic space with absent or attenuated myoepithelial cell layer. Although staged as an in-situ lesion, it can be associated with invasive ductal carcinoma (IDC). We sought to compare the genomic characteristics of pure EPC and EPC with associated invasive carcinoma (EPCi) at the genomic level. All cases of EPCi harbored recurrent hotspot mutations in PIK3CA. PIK3CA, KMT2A and CREBBP deleterious somatic events were found across both tumor groups, irrespective of invasion status. At the whole transcriptomic level, EPCi cases displayed remarkably similar mRNA profiles when compared to EPC. When EPCi cases were compared with their corresponding IDC, despite significant overlap, we identified differential gene expression in 39 genes with enrichment of multiple pathways including extracellular matrix regulation, cell adhesion and collagen fibril organization. Despite morphologic, genotypic and transcriptomic overlap between pure EPC and EPCi, the latter tumors are likely advanced lesions with PIK3CA activating mutations and enrichment of stromal-related genes implicated in the switch to IDC.

Recent genomic studies have shed light on the biology and inter-tumoral heterogeneity underlying pineal parenchymal tumors, in particular pineoblastomas (PBs) and pineal parenchymal tumors of intermediate differentiation (PPTIDs). Previous reports, however, had modest sample sizes and lacked the power to integrate molecular and clinical findings. The different proposed molecular group structures also highlighted a need to reach consensus on a robust and relevant classification system. We performed a meta-analysis on 221 patients with molecularly characterized PBs and PPTIDs. DNA methylation profiles were analyzed through complementary bioinformatic approaches and molecular subgrouping was harmonized. Demographic, clinical, and genomic features of patients and samples from these pineal tumor groups were annotated. Four clinically and biologically relevant consensus PB groups were defined: PB-miRNA1 (n = 96), PB-miRNA2 (n = 23), PB-MYC/FOXR2 (n = 34), and PB-RB1 (n = 25). A final molecularly distinct group, designated PPTID (n = 43), comprised histological PPTID and PBs. Genomic and transcriptomic profiling allowed the characterization of oncogenic drivers for individual tumor groups, specifically, alterations in the microRNA processing pathway in PB-miRNA1/2, MYC amplification and FOXR2 overexpression in PB-MYC/FOXR2, RB1 alteration in PB-RB1, and KBTBD4 insertion in PPTID. Age at diagnosis, sex predilection, and metastatic status varied significantly among tumor groups. While patients with PB-miRNA2 and PPTID had superior outcome, survival was intermediate for patients with PB-miRNA1, and dismal for those with PB-MYC/FOXR2 or PB-RB1. Reduced-dose CSI was adequate for patients with average-risk, PB-miRNA1/2 disease. We systematically interrogated the clinical and molecular heterogeneity within pineal parenchymal tumors and proposed a consensus nomenclature for disease groups, laying the groundwork for future studies as well as routine use in tumor diagnostic classification and clinical trial stratification.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications.