Faculty Bibliography

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

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: 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

Medulloblastoma (MB) is the most common childhood malignant brain tumor and is a leading cause of cancer-related death in children. DNA methylation profiling has rapidly advanced our understanding of MB pathogenesis at the molecular level, but assessments in Saudi Arabian (SA)-MB cases are sparse. MBs can be sub-grouped according to methylation patterns from FPPE samples into Wingless (WNT-MB), Sonic Hedgehog (SHH-MB), Group 3 (G3), and Group 4 (G4) tumors. The WNT-MB and SHH-MB subgroups are characterized by gain-of function mutations that activate oncogenic cell signaling, whilst G3/G4 tumors show recurrent chromosomal alterations. Given that each subgroup has distinct clinical outcomes, the ability to subgroup SA-FPPE samples holds significant prognostic and therapeutic value. Here, we performed the first assessment of MB-DNA methylation patterns in an SA cohort using archival biopsy material (FPPE n = 49). Of the 41 materials available for methylation assessments, 39 could be classified into the major DNA methylation subgroups (SHH, WNT, G3, and G4). Furthermore, methylation analysis was able to reclassify tumors that could not be sub-grouped through next-generation sequencing, highlighting its superior accuracy for MB molecular classifications. Independent assessments demonstrated known clinical relationships of the subgroups, exemplified by the high survival rates observed for WNT tumors. Surprisingly, the G4 subgroup did not conform to previously identified phenotypes, with a high prevalence in females, high metastatic rates, and a large number of tumor-associated deaths. Taking our results together, we demonstrate that DNA methylation profiling enables the robust sub-classification of four disease sub-groups in archival FFPE biopsy material from SA-MB patients. Moreover, we show that the incorporation of DNA methylation biomarkers can significantly improve current disease-risk stratification schemes, particularly concerning the identification of aggressive G4 tumors. These findings have important implications for future clinical disease management in MB cases across the Arab world.

Glioblastoma, an incurable tumor, remains difficult to model and more importantly to treat due to its genetic/epigenetic heterogeneity and plasticity across cellular states. The ability of current tumor models to recapitulate the cellular states found in primary tumors remains unexplored. To address this issue, we compared single-cell RNA-sequencing of tumor cells from five patients across four patient-specific glioblastoma stem cell (GSC)-derived model types, including glioma spheres, tumor organoids, glioblastoma cerebral organoids (GLICO), and patient-derived xenografts. We find that GSCs within the GLICO model are enriched for a neural progenitor-like cell (NPC) subpopulation and recapitulate the cellular states and their plasticity found in the corresponding primary parental tumors. These data demonstrate how the contribution of a neuroanatomically accurate human microenvironment is critical and sufficient for recapitulating the cellular states found in human primary GBMs, a principle that may likely apply to other tumor models.

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.

Infant high grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histological review, methylation profiling, custom panel and genome/exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an 'intrinsic' spectrum of disease specific to the infant population. These included those with targetable MAP-kinase alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n=31), NTRK1/2/3 (n=21), ROS1 (n=9) and MET (n=4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly supports the concept that infant gliomas require a change in diagnostic practice and management.

BACKGROUND:Both genetic and methylation analysis have been shown to provide insight into the diagnosis and prognosis of many brain tumors. However, the implication of methylation profiling and its interaction with genetic alterations in pediatric low-grade gliomas (PLGGs) are unclear. METHODS:We performed a comprehensive analysis of PLGG with long term clinical follow up. In total 152 PLGGs were analyzed from a range of pathological subtypes including 40 gangliogliomas. Complete molecular analysis was compared to genome-wide methylation data and outcome in all patients. For further analysis of specific PLGG groups, including BRAF p.V600E mutant gliomas, we compiled an additional cohort of clinically and genetically defined tumors from 3 large centers. RESULTS:Unsupervised hierarchical clustering revealed 5 novel subgroups of PLGG. These were dominated by non-neoplastic factors such as tumor location and lymphocytic infiltration. Midline PLGG clustered while deep hemispheric lesions differed from lesions in the periphery. Mutations were distributed throughout these location-driven clusters of PLGG. A novel methylation cluster suggesting high lymphocyte infiltration was confirmed pathologically and exhibited worse progression-free survival compared to PLGG harboring similar molecular alterations (p = 0.008, multivariate analysis: p = 0.035). Although the current methylation classifier revealed low confidence in 44% of cases and failed to add information in most PLGG it was helpful in reclassifying rare cases. The addition of histopathological and molecular information to specific methylation subgroups such as pleomorphic xanthoastrocytoma (PXA)-like tumors could stratify these tumors into low and high risk (p = 0.0014). CONCLUSION/CONCLUSIONS:The PLGG methylome is affected by multiple non-neoplastic factors. Combined molecular and pathological analysis is key to provide additional information when methylation classification is used for PLGG in the clinical setting.

Of the four primary subgroups of medulloblastoma, the most frequent cytogenetic abnormality, i17q, distinguishes Groups 3 and 4 which carry the highest mortality; haploinsufficiency of 17p13.3 is a marker for particularly poor prognosis. At the terminal end of this locus lies miR-1253, a brain-enriched microRNA that regulates bone morphogenic proteins during cerebellar development. We hypothesized miR-1253 confers novel tumor-suppressive properties in medulloblastoma. Using two different cohorts of medulloblastoma samples, we first studied the expression and methylation profiles of miR-1253. We then explored the anti-tumorigenic properties of miR-1253 in parallel with a biochemical analysis of apoptosis and proliferation, and isolation of oncogenic targets using high-throughput screening. Deregulation of miR-1253 expression was noted, both in medulloblastoma clinical samples and cell lines, by epigenetic silencing via hypermethylation; specific de-methylation of miR-1253 not only resulted in rapid recovery of expression but also a sharp decline in tumor cell proliferation and target gene expression. Expression restoration also led to a reduction in tumor cell virulence, concomitant with activation of apoptotic pathways, cell cycle arrest, and reduction of markers of proliferation. We identified two oncogenic targets of miR-1253, CDK6 and CD276, whose silencing replicated the negative trophic effects of miR-1253. These data reveal novel tumor-suppressive properties for miR-1253, i.e. 1) loss of expression via epigenetic silencing, 2) negative trophic effects on tumor aggressiveness, and 3) downregulation of oncogenic targets.

Gene fusions are caused by chromosomal rearrangements and encode fusion proteins that can act as oncogenic drivers in cancers. Traditional methods for detecting oncogenic fusion transcripts include fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). However, these methods are limited in scalability and pose significant technical and interpretational challenges. Next-generation sequencing (NGS) is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for cancer patients. We present our experience with the validation of a custom-designed Archer Anchored Multiplex PCR (AMP™) technology-based NGS technology, "NYU FUSION-SEQer" using RNA sequencing. We examine both analytical performance and clinical utility of the panel using 75 retrospective validation samples and 84 prospective clinical samples of solid tumors. Our panel showed robust sequencing performance with strong enrichment for target regions. The lower limit of detection was 12.5% tumor fraction at 125 ng of RNA input. The panel demonstrated excellent analytic accuracy, with 100% sensitivity, 100% specificity and 100% reproducibility on validation samples. Finally, in the prospective cohort, the panel detected fusions in 61% cases (n = 51), out of which 41% (n = 21) enabling diagnosis and 59% (n = 30) enabling treatment and prognosis. We demonstrate that the fusion panel can accurately, efficiently and cost-effectively detect the majority of known fusion genes, novel clinically relevant fusions and provides an excellent tool for discovery of new fusion genes in solid tumors.