Faculty Bibliography

Background/UNASSIGNED:Glioma is a family of primary brain malignancies with limited treatment options and in need of novel therapies. We previously demonstrated that the adhesion G protein-coupled receptor GPR133 (ADGRD1) is necessary for tumor growth in adult glioblastoma, the most advanced malignancy within the glioma family. However, the expression pattern of GPR133 in other types of adult glioma is unknown. Methods/UNASSIGNED:We used immunohistochemistry in tumor specimens and non-neoplastic cadaveric brain tissue to profile GPR133 expression in adult gliomas. Results/UNASSIGNED:We show that GPR133 expression increases as a function of WHO grade and peaks in glioblastoma, where all tumors ubiquitously express it. Importantly, GPR133 is expressed within the tumor bulk, as well as in the brain-infiltrating tumor margin. Furthermore, GPR133 is expressed in both isocitrate dehydrogenase (IDH) wild-type and mutant gliomas, albeit at higher levels in IDH wild-type tumors. Conclusion/UNASSIGNED:The fact that GPR133 is absent from non-neoplastic brain tissue but de novo expressed in glioma suggests that it may be exploited therapeutically.

Pleomorphic xanthoastrocytoma (PXA) is a rare type of brain tumor that affects children and young adults. Molecular prognostic markers of PXAs remain poorly established. Similar to gangliogliomas, PXAs show prominent immune cell infiltrate, but its composition also remains unknown. In this study, we correlated DNA methylation and BRAF status with clinical outcome and explored the tumor microenvironment. We performed DNA methylation in 21 tumor samples from 18 subjects with a histological diagnosis of PXA. MethylCIBERSORT was used to deconvolute the PXA microenvironment by analyzing the associated immune cell-types. Median age at diagnosis was 16 years (range 7-32). At median follow-up of 30 months, 3-year and 5-year overall survival was 73% and 71%, respectively. Overall survival ranged from 1 to 139 months. Eleven out of 18 subjects (61%) showed disease progression. Progression-free survival ranged from 1 to 89 months. Trisomy 7 and CDKN2A/B (p16) homozygous deletion did not show any association with overall survival (p = 0.67 and p = 0.74, respectively). Decreased overall survival was observed for subjects with tumors lacking the BRAF V600E mutation (p = 0.02). PXAs had significantly increased CD8 T-cell epigenetic signatures compared with previously profiled gangliogliomas (p = 0.0019). The characterization of immune cell-types in PXAs may have implications for future development of immunotherapy.

A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacological inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggests that PHGDH inhibitors may be useful in the treatment of brain metastasis.

Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epidemiological parameters. Here, we report the analysis of 236 SARS-CoV2 sequences from cases in the New York City metropolitan area during the initial stages of the 2020 COVID-19 outbreak. The majority of cases throughout the region had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that the majority were most related to cases from Europe. Our data are consistent with numerous seed transmissions from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of real-time genomic surveillance in addition to traditional epidemiological indicators.

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

Background: Clear cell papillary renal cell carcinoma (ccpRCC) is a relatively new entity and it has been described as an indolent renal neoplasm. ccpRCC shares features of clear cell RCC (ccRCC) and papillary RCC (pRCC) morphologically and immunohistochemically, even though it carries a very different prognostic potential. Epigenetic alterations play a significant role in the development and progression of human tumors. A large scale sequencing efforts demonstrated that hypermethylation in RCC tumors is associated with poor prognosis and may dictate treatment options. However, ccpRCC has not been included and further molecular elucidation is to be done. In this study, we attempt to investigate the methylation patterns in ccpRCC and test if differential patterns can be correlated with histologic subtypes and known biological behaviors.
Design(s): Nephrectomy specimens from our institution were reviewed and 8 cases from 2017-2019 were selected and confirmed as ccpRCC by three pathologists. Tumors were microdissected and DNA from FFPE was extracted and profiled using the 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 minimum mean difference of the Beta-value of 0.1. Clustering was performed using tSNE analysis. Copy numbers were analyzed using conumee package. The methylation data were compared to ccRCC and pRCC from publicly available TCGA dataset.
Result(s): All 8 cases passed QC metrics on bisulfite conversion, hybridization and signal intensity confirming that the DNA quality was optimal for methylation study. ccpRCC clustered very tightly together illustrating that they represented a homogeneous group of tumors. When this cluster was compared to the TCGA dataset, ccpRCC was found to be located in between ccRCC and pRCC which might explain the characteristic features of this tumor subtype (figure 1). (Figure presented)
Conclusion(s): 1. DNA methylation is a useful molecular hallmark of many cancers including renal cancers and is increasingly utilized in diagnosis, prognostication, and clinical trials (epigenetic therapy). 2. Morphologically and immunohistochemically confirmed ccpRCC forms a tight cluster validating the use of methylation assay for future studies. 3. Promotor and pathway specific methylation patterns will be further studied which can distinguish the indolent clinical behavior

Background: Encapsulated papillary carcinomas (EPC) of the breast is a variant of papillary carcinoma that are confined to a cystic space, surrounded by a fibrous capsule and lack the myoepithelial coat. Despite the latter finding, it is recommended that EPC be staged as pTis due to its indolent course. Concurrent frank invasive carcinomas are staged commensurate with their size. We sought to investigate the molecular pathways differentially expressed in pure EPC and EPC with frank invasion at the genomic and transcriptomic level. In addition, we compared EPC with its corresponding invasive ductal carcinoma (IDC) at the transcriptomic level.
Design(s): We selected 3 cases of pure EPC (C1-C3) and 3 cases of EPC (C4e-C6e) with corresponding IDC (C4i-C6i).We performed whole transcriptome analysis on laser-capture microdissected samples from formalin-fixed, paraffin-embedded tissue. We used CloneTech Mammalian stranded pico kit for sequencing RNA. KEGG pathway analysis and Gene Ontology (GO) analysis was performed using the cluster Profiler R package (v3.0.0) and Database for Annotation, Visualization and Integrated Discovery. DNA analysis was performed using our in-house next generation sequencing hybrid capture covering 580 genes on C1-C3 and C4e-C6e.
Result(s): There were 5 female and 1 male patients. The mean age was 73 years (range 62-90). All cases were hormone receptor positive. C4e-C6e showed upregulation of NTRK2 and MAGI2 (lg2FC= 3.14 and lg2FC = 3.0 fold, respectively) and downregulation of PRKACB (lg2FC= -4.4) compared to C1-C3 on RNAseq. C4i-C6i showed upregulation of collagen-related genes (COL10A1, COL11A1, COL14A1, COL16A1, COL1A1, COL3A1, COL8A1) (lg2FC range: 6.28 fold change) and ADAM12/ADAMTS2 (lg2FC=6.2 and lg2FC= 6.9 fold change) compared to C4e-C6e (FDR =0.014). Pathway analysis showed upregulation of collagen fibril organization and extracellular matrix organization pathways in C4i-C6i compared to C4e-C6e and upregulation of kinase activity pathway (GO: 0016301) in C4e-C6e compared to C1-C3.Recurrent PIK3CA hotspot non-synonymous mutation was identified in C3, C4e, C5e and C6e (c.G1633A in C5 and c.A3140G in C3, C4 and C6).
Conclusion(s): Our findings suggest that kinase and matrix metalloproteinase pathways contribute to EPC with invasion compared to pure EPC cases. Furthermore, enrichment of collagen-related genes in IDCs compared to their corresponding EPC suggest a synergistic potential with the aforementioned pathways. Mechanistic studies are warranted to validate the findings

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: As hepatocellular carcinoma (HCC) develops from premalignant lesions (low and high grade dysplastic nodules; LGDN and HGDN), there is a corresponding accumulation of molecular alterations, some of which have been well described. However, the molecular features of lesions comprising the hepatocellular dysplasia-carcinoma sequence as they relate to different etiologies have not yet been explored. Herein, we characterize the molecular landscape of such lesions in cirrhotic explants with alcohol-related liver disease (ALD) and chronic hepatitis C (CHC).
Design(s): Tissue was assessed from 27 liver explants (14 CHC, 13 ALD) including 10 LGDNs (5 CHC, 5 ALD), 8 HGDNs (3 CHC, 5 ALD), 10 HCCs arising from HGDNs (5 CHC, 5 ALD), and 10 small HCCs defined as HCC < 2 cm (5 CHC, 5 ALD), as well as non-lesional cirrhotic parenchyma and matched normal non-liver tissue (e.g. porta hepatis structures). DNA was extracted from FFPE tissue for next generation sequencing (NGS) using a customized NGS580 panel targeting all exonic and select intronic areas in 580 cancer related genes. Data was analyzed using customized bioinformatics pipelines with an R package.
Result(s): TERT promoter HS C228T mutations were identified in 6 of 10 (60%) CHC related HCCs and only 1 of 9 (11%) alcohol related HCC (Figure 1). There was a significant association between TERT promoter HS C228T mutations and CHC related HCCs (p<0.02). In contrast, ALD related lesions showed deleterious events affecting tumor suppressor genes (NF1, BRCA1; CDKN2C) and histone methylation/chromatin remodeling genes (KMT2A; KMT2C; ASXL1; RAD21), which were found in 6 of 13 ALD related lesions (46.2% [3 of 5 small HCCs, 2 of 4 HGDNs, and 1 of 4 HCCs arising in HGDNs]). These events only occurred in 3 of 14 (21.4%) CHC related lesions (Figures 1 and 2). Within the CHC group, 1 HCC arising in HGDN showed copy number gains (CNG) in MARK4, ERCC2, FGFR4 and FLT4 and two HGDNs showed CNGs in NOTCH1 and TERT, respectively. No differences in the tumor mutational burden (TMB) were noted between CHC related and ALD related lesions, nor across the DN-HCC sequence. Non-lesional liver and LGDNs did not show recurrent mutations pertaining to a specific pathway. (Figure presented)
Conclusion(s): Our findings suggest that the pathways of hepatocarcinogenesis are distinct in ALD and CHC. While upregulation of telomerase activity (TA) and cancer cell immortalization play a pivotal role in CHC related HCC, defective chromatin remodeling appears to contribute to tumorigenesis in ALD related HCC

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.