Faculty Bibliography

Screening for therapeutic targets is standard of care in the management of advanced non-small cell lung cancer. However, most molecular assays utilize tumor tissue, which may not always be available. "Liquid biopsies" are plasma-based next generation sequencing (NGS) assays that use circulating tumor DNA to identify relevant targets. To compare the sensitivity, specificity, and accuracy of a plasma-based NGS assay to solid-tumor-based NGS we retrospectively analyzed sequencing results of 100 sequential patients with lung adenocarcinoma at our institution who had received concurrent testing with both a solid-tissue-based NGS assay and a commercially available plasma-based NGS assay. Patients represented both new diagnoses (79%) and disease progression on treatment (21%); the majority (83%) had stage IV disease. Tissue-NGS identified 74 clinically relevant mutations, including 52 therapeutic targets, a sensitivity of 94.8%, while plasma-NGS identified 41 clinically relevant mutations, a sensitivity of 52.6% (p < 0.001). Tissue-NGS showed significantly higher sensitivity and accuracy across multiple patient subgroups, both in newly diagnosed and treated patients, as well as in metastatic and nonmetastatic disease. Discrepant cases involved hotspot mutations and actionable fusions including those in EGFR, ALK, and NTRK1. In summary, tissue-NGS detects significantly more clinically relevant alterations and therapeutic targets compared to plasma-NGS, suggesting that tissue-NGS should be the preferred method for molecular testing of lung adenocarcinoma when tissue is available. Plasma-NGS can still play an important role when tissue testing is not possible. However, given its low sensitivity, a negative result should be confirmed with a tissue-based assay.

Gliomas are the most common adult and pediatric primary brain tumors. Molecular studies have identified features that can enhance diagnosis and provide biomarkers. IDH1/2 mutation with ATRX and TP53 mutations defines diffuse astrocytomas, whereas IDH1/2 mutations with 1p19q loss defines oligodendroglioma. Focal amplifications of receptor tyrosine kinase genes, TERT promoter mutation, and loss of chromosomes 10 and 13 with trisomy of chromosome 7 are characteristic features of glioblastoma and can be used for diagnosis. BRAF gene fusions and mutations in low-grade gliomas and histone H3 mutations in high-grade gliomas also can be used for diagnostics.

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.

Glioblastoma (GBM) is the most common malignant primary central nervous system (CNS) neoplasm in adults, and has an almost universally poor prognosis. Recently, an emphasis on genetic and epigenetic profiling has revealed a number of molecular features useful in the diagnostic and prognostic classification of GBM, advancing our understanding of the underlying features that make these tumors so aggressive and providing the rationale for the creation of better targeted therapeutics. One such method, DNA methylation profiling, has recently emerged as an important technique for the classification of CNS tumors, with diagnostic accuracy in some cases surpassing traditional methods. However, how DNA methylation profiles change with the course of the disease remains less understood. Here, we present a case of a 30-year-old male with primary IDH-mutant GBM with widespread recurrence and death two years later. Using unsupervised hierarchical clustering of methylation probes, we created a phylogenetic map to trace the tumor path as it spread from the initial biopsy site throughout the right hemisphere, across the corpus callosum to the contralateral hemisphere, and into the brainstem. We identified molecular divergence between the right and left hemisphere GBM samples marked by distinct copy number profile alterations, alterations in specific methylation sites, and regional loss of MGMT promoter methylation, providing a potential mechanism for treatment resistance in this case. In summary, this case both highlights the molecular diversity in GBM, and illustrates a novel use for methylation profiling in establishing a phylogenetic profile to allow for spatial mapping of tumor progression.

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.