Faculty Bibliography

BACKGROUND: Tumor heterogeneity presents a major challenge to cancer diagnosis and treatment. In addition to interpatient tumor variability, intratumoral heterogeneity characterized by distinct molecular and phenotypic profiles is increasingly recognized as a major cause of therapy resistance and cancer recurrence. Because DNA methylation patterns are largely responsible for determining cell-type-specific functioning, we hypothesized that distinct DNA methylation and proteomic alterations could be identified in histologically-defined invasive and proliferative tumor cell populations in human isocitrate dehydrogenase 1 (IDH1)- mutated and wild-type glioblastoma (GBM).
METHOD(S): Formalin-fixed paraffin-embedded tissue sections of human adult IDH1-mutated and wild-type GBM were laser-microdissected (LM) into perinecrotic pseudopalisading tumor cells (PPCs), non-pseudopalisading tumor core cells (NPPCs), invasive subpial spread (SPS) and perivascular satellitosis tumor cells and brain adjacent to tumor cells prior to analysis and compared to non-microdissected tumor (NMT) and/or germline DNA. Genomewide DNA methylation and chromosomal copy numbers were determined with Infinium MethylationEPIC 850K BeadChip and intratumoral DNA methylation patterns compared by unsupervised hierarchical clustering. Label-free quantitative liquid chromatography-mass spectrometry of proteins was performed and proteins differentially expressed across LM areas subjected to pathway enrichment analysis.
RESULT(S): Unsupervised hierarchical classification of DNA methylation patterns for each LM area and NMT demonstrated remarkable clustering for all patients, based on methylation probe and methylated gene patterns. Proteomics analysis showed upregulation of hypoxia-inducible factor-1 inducible proteins in hypoxic PPCs. Out of 1819 proteins quantified, 5 were overexpressed and 9 underexpressed more than 10-fold in SPS compared with NPPCs and associated with alterations in metabolism, transport, extracellular matrix and apoptosis. Correlation of protein expression and DNA methylation patterns was noted.
CONCLUSION(S): Compared to NPPCs, SPS cells migrating toward the invasive edge share a relatively consistent epigenetic and proteomic signature, suggesting potentially targetable common mechanism(s) of invasion shared among GBM

A methionine substitution at lysine-27 on histone H3 variants (H3K27M) characterizes ~80% of diffuse intrinsic pontine gliomas (DIPG) and inhibits polycomb repressive complex 2 (PRC2) in a dominant-negative fashion. Yet, the mechanisms for this inhibition and abnormal epigenomic landscape have not been resolved. Using quantitative proteomics, we discovered that robust PRC2 inhibition requires levels of H3K27M greatly exceeding those of PRC2, seen in DIPG. While PRC2 inhibition requires interaction with H3K27M, we found that this interaction on chromatin is transient, with PRC2 largely being released from H3K27M. Unexpectedly, inhibition persisted even after PRC2 dissociated from H3K27M-containing chromatin, suggesting a lasting impact on PRC2. Furthermore, allosterically activated PRC2 is particularly sensitive to H3K27M, leading to the failure to spread H3K27me from PRC2 recruitment sites and consequently abrogating PRC2's ability to establish H3K27me2-3 repressive chromatin domains. In turn, levels of polycomb antagonists such as H3K36me2 are elevated, suggesting a more global, downstream effect on the epigenome. Together, these findings reveal the conditions required for H3K27M-mediated PRC2 inhibition and reconcile seemingly paradoxical effects of H3K27M on PRC2 recruitment and activity.

Visual inspection of histopathology slides is one of the main methods used by pathologists to assess the stage, type and subtype of lung tumors. Adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are the most prevalent subtypes of lung cancer, and their distinction requires visual inspection by an experienced pathologist. In this study, we trained a deep convolutional neural network (inception v3) on whole-slide images obtained from The Cancer Genome Atlas to accurately and automatically classify them into LUAD, LUSC or normal lung tissue. The performance of our method is comparable to that of pathologists, with an average area under the curve (AUC) of 0.97. Our model was validated on independent datasets of frozen tissues, formalin-fixed paraffin-embedded tissues and biopsies. Furthermore, we trained the network to predict the ten most commonly mutated genes in LUAD. We found that six of them-STK11, EGFR, FAT1, SETBP1, KRAS and TP53-can be predicted from pathology images, with AUCs from 0.733 to 0.856 as measured on a held-out population. These findings suggest that deep-learning models can assist pathologists in the detection of cancer subtype or gene mutations. Our approach can be applied to any cancer type, and the code is available at https://github.com/ncoudray/DeepPATH .

BACKGROUND AND PURPOSE/OBJECTIVE:Few studies have shown MR imaging features and ADC correlating with molecular markers and survival in patients with glioma. Our purpose was to correlate MR imaging features and ADC with molecular subtyping and survival in adult diffuse gliomas. MATERIALS AND METHODS/METHODS:promoter methylation, and overall survival. RESULTS:wild-type glioma. Other MR imaging features were not statistically significant predictors of survival. CONCLUSIONS:wild-type gliomas.

In the version of this Letter originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Letter.

CNS-PNET is no longer regarded a single disease but encompassed many distinct molecular entities. After removal of the term from the 2016 WHO classification of CNS tumours, diagnostic and therapeutic uncertainty remains. Through a world-wide collaboration, tumour samples from patients with the "historic" diagnosis of CNS-PNET were re-evaluated by DNA methylation profiling (n=405) and blinded neuropathological panel review (n=256). Clinical data on treatment and outcome were pooled with data on previously published patients. The given numbers represent preliminary data of the ongoing project. The re-evaluation by DNA methylation identified many distinct entities as expected, including high grade glioma (HGG, n=70), embryonal tumors with multilayered rosettes (ETMR, n=57) and CNS-neuroblastoma with FOXR2 alteration (CNS-NB-FOXR2, n=42) as the most frequent molecular diagnostic categories. Poor clinical outcome was confirmed for patients with HGG (5y-PFS 12%/5y-OS 12%, n=24), and ETMR (5y-PFS 12%/5y-OS 18%, n=62), while most patients with CNS-NBFOXR2 survived (5y-PFS 52%/5y-OS 96%, n=31). Seven of 12 relapses/progressions of CNS-NB-FOXR2 occurred in radiotherapy-naive patients. Classification into other newly described and less common entities included HGNET-MN1 (n=19), HGNET-BCOR (n=11), and EFT-CIC (n=13). Independent neuropathological review demonstrated that samples of these entities presented as non-embryonal tumours. Furthermore, marked clinical differences exist (HGNET-MN1: 5y-PFS 25%/5y-OS 95%, n=22; HGNETBCOR: 5y-PFS 0%/5y-OS 44%, n=16; CNS-EFT-CIC: 5y-PFS 40%/5y-OS 60%, n=10). Our results show that implementation of DNA methylation profiling together with histopathological analysis will improve prospective diagnostic accuracy and facilitates the retrospective outcome analysis of treatment protocols used for this variety of biologically distinct entities

Several different entities with distinct clinical and histopathological features are described within in the group of pineal tumors. Whereas pineocytoma (PC), pineal parenchymal tumors of intermediate differentiation (PPTID) and papillary tumors of the pineal region (PTPR) predominantly affect adults and are associated with a relatively favorable prognosis, pineoblastoma (PB) occurs at young age and constitutes a highly aggressive tumor. Despite multimodal treatment, prognosis is poor (especially for infants and patients with metastatic disease), and therapeutically actionable molecular targets are lacking to date. Most PB arise sporadically, however, DICER1 or RB1 germline mutations are known to predispose to the development of PB, the latter in the context of trilateral retinoblastoma. Genome-wide DNA methylation profiling and copy-number analysis were used to investigate the biological features of 230 pineal tumors of different histologies. Known histopathological entities (PC, PTPR, PPTID) could be clearly separated by unsupervised clustering, as well as further distinct subgroups, including the previously hypothesized PTPR-A and PTPR-B groups. Notably, several biologically discrete subgroups were formed by the tumors initially diagnosed as PB or pineal primitive embryonal tumors/PNETs, which showed distinct clinical associations (e.g. age distribution). Somatic deletions of DROSHA, an endoribonuclease involved in miRNA processing upstream of DICER1, seem to be a recurrent feature of the largest subgroup. Rarer DICER1 and DGCR8 alterations in this group confirm a central role of altered miRNA biogenesis in the development of this subset of PB. Further molecular and functional characterization of novel sub-clusters, including ongoing miRNAseq, will provide insights into the oncogenesis of PB

BACKGROUND: Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. METHODS: We analyzed pediatric and adult pineoblastoma samples (n=23) using integrated genomic studies, including genome-wide DNA methylation profiling, whole-exome or whole-genome sequencing, and whole-transcriptome analysis. RESULTS: Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower grade pineal tumors and normal pineal gland. Recurrent somatic mutations were found in genes involved in PKA-and NF-kappaB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expression of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain. Whole-transcriptome analysis showed that homozygous loss of DROSHA led to distinct changes in RNA expression profile. Disruption of the DROSHA locus in human neural stem cells using the CRISPR/Cas9 system, led to decrease of the DROSHA protein, and massive loss of miRNAs. CONCLUSION: We identified recurrent homozygous deletions of DROSHA in pineoblastoma, suggesting that different mechanisms disrupting miRNA processing are involved in the pathogenesis of familial versus sporadic pineoblastoma. Furthermore, a novel microduplication of PDE4DIP leading to upregulation of DUF1220 protein suggests DUF1220 as a novel oncogenic driver in pineoblastoma