TCGA Classification Research Articles

Molecular classification and subtype-specific characterization of skin cutaneous melanoma by aggregating multiple genomic platform data.

Traditional classification of melanoma is widely utilized with little apparent results making the development of robust classifiers that can guide therapies an urgency. Successful seminal research on classification has provided a wider understanding of cancer from multiple molecular profiles, respectively. However, it may ignore the complementary nature of the information provided by different types of data, which motivated us to subtype melanoma by aggregating multiple genomic platform data. Aggregating three omics data of 328 melanoma samples, melanoma subtyping was performed by three clustering methods. Differences across subtypes were extracted by functional enrichment, epigenetically silencing, gene mutations and clinical features. Subtypes were further distinguished by putative biomarkers. Functional enrichment of the subtype-specific differential expression genes endowed subtypes new designation: immune, melanin and ion, in which the first subtype was enriched for immune system, the second was characterized by melanin and pigmentation, and the third was enriched for ion-involved transmission process. Subtypes also differed in age, Breslow thickness, tumor site, mutation frequency of BRAF, PTGS2, CDKN2A, CDKN2B and incidence of epigenetically silencing for IL15RA, EPSTI1, LXN, CDKN1B genes. Skin cutaneous melanoma can be robustly divided into three subtypes by SNFCC+. Compared with the TCGA classification derived from gene expression, the subtypes we presented share concordance, but new traits are excavated. Such a genomic classification offers insights to further personalize therapeutic decision-making and melanoma management.

PO-214 Conditional control of MITF reveals cellular subpopulations essential for melanoma survival and recurrence in new zebrafish models

Introduction Melanoma is the most lethal type of skin cancer and its incidence continues to rise rapidly. While BRAFV600E inhibitors have led to significant improvements in outcomes for some patients, there are no targeted therapies available for melanoma patients who lack BRAF mutations. An emerging area of new melanoma targets is the melanocyte lineage, which is critical for melanoma survival and contributes to therapeutic resistance. MITF is a conserved ‘master melanocyte regulator’ with a complex role in melanoma. Our lab previously developed a BRAFV600E mitf zebrafish melanoma model carrying a human oncogene and mitfvc7 splice site mutation that enables the conditional control of its endogenous activity. Since there are not many adequate animal models to study the non-BRAF and non-RAS melanomas, we have now also developed a double mutant model p53 mitf. Here we use the new models to investigate MITF-dependent melanoma regression and recurrence. Material and methods We use histopathology and imaging of melanocyte lineage fluorescent reporters (mitf and crestin) in new temperature-sensitive zebrafish models, as well as primary zebrafish melanoma cell line, to study melanoma regression and recurrence. Results and discussions We demonstrate that the MITF activity is crucial for melanocyte survival and that both mutated BRAF and p53 deficiency are oncogenic with low levels of MITF, and result in melanoma resembling the pathology of human disease. This is particularly important considering the new MITF-low subclass of human melanomas that has emerged from the recent TCGA classification. We also show that complete inhibition of MITF activity leads to rapid tumour regression (in both new models), but once its activity is restored the melanomas recur at the same site as the original tumour. This suggests that a subpopulation of cancer initiating cells remains following melanoma regression and is capable of repopulating the tumour. We have been able to show that some cells remaining at the sites of regression express MITF and/or ALDH2 and we are currently searching for other markers. Conclusion We developed new MITF-dependent zebrafish models of melanoma that are relevant to distinct clinical subtypes. We showed that inhibiting MITF in those animals results in melanoma regression, but is not sufficient to eradicate the tumours (once its activity is restored, melanomas recur). We identified subpopulations of cells remaining at the regression sites and are investigating the nature of these proposed cells-of-origin for relapse.

308 EGFR Gene Amplification Predicts a Poor Response to Bevacizumab in Recurrent Glioblastoma

Abstract INTRODUCTION The highly vascular malignant brain tumor glioblastoma multiforme (GBM) appears to be an ideal target for anti-angiogenic therapy; however, clinical trials to date suggest the VEGF antibody bevacizumab affects only progression free survival without any benefit on overall survival. Here we analyze a group of patients with GBM who received bevacizumab treatment and are stratified according to tumor molecular and genomic profile (TCGA classification), with the goal of identifying molecular predictors of the response to bevacizumab. METHODS We performed a retrospective review of patients with a diagnosis of glioblastoma multiforme who were treated with bevacizumab in the recurrent setting at Memorial Sloan-Kettering Cancer Center, from 2006 to 2014. Treatment was discontinued by individual neuro-oncologists, based on clinical and radiographic criteria. Pre- and post- treatment imaging and genomic subtype were available on 83 patients; We analyzed clinical progression, and time to radiographic progression (rP) on MRI flair and T1 weighted images with contrast, quantified tumor volumes as well as the development of multifocal lesions. EGFR gene amplification and MGMT promotor methylation status were determined by FISH. RESULTS >EGFR gene amplification was significantly associated with a shorter time to progression both in univariate (P = < 0.001) and multivariate analysis (P = 0.011). Patients with classical tumors had a significantly shorter time to progression than mesenchymal and proneural patients (2.5 vs 6.32 and 5.2 months respectively, P = 0.001), although it lost its significance (P = 0.262) on multivariate analysis when correcting for EGFR status. Overall survival was significantly shorter in patients with EGFR- amplified GBM treated with bevacizumab. CONCLUSION EGFR gene amplification and classical subtype by TCGA analysis are associated with significantly shorter time to progression for patients with recurrent GBM when treated with bevacizumab. These findings can have a significant impact on decision making and should be further validated prospectively.

Abstract 3559: Functional characterization of HNF4α in gastric cancer

Abstract Gastric cancer (GC) is a leading cause of cancer mortality worldwide and particularly common in East Asia. Transcription factors are frequently amplified or overexpressed in GC and might contribute to tumorgenesis, however the underlying mechanisms largely remain unknown. Our previous data indicates that HNF4α is a common target of three amplified transcription factors (GATA4, GATA6 and KLF5) in gastric cancer. HNF4α had also been found to be related to diverse aspects of tumorigenesis. In liver and colon cancers, there are reports indicating that the targets of HNF4α are mainly involved in intermediary metabolism, however, its tissue-specific downstream targets and pathways have not been systemically studied yet. Therefore, a comprehensive functional characterization of HNF4α is required to identify its tissue-specific role in gastric cancer. In this study, we analyzed the expression features of HNF4α in GC, and found the expression level of HNF4α is significantly higher in early stage tumor and in CIN and MSI compared to GS and EBV subtypes of GC according to TCGA classification method. In addition, we performed the HNF4α ChIP in GC cell lines, and the results revealed a number of HNF4α binding sites in GC. Comparison with the published ChIP-seq data of Caco2 and HepG2 cell lines indicated the existence of GC-exclusive targets. Furthermore, functional annotation analysis revealed that the GC-exclusive targets are significantly related to the negative regulation of cellular metabolic process compared to the common targets. These preliminary data suggests that HNF4α might play a tissue specific role in GC, especially in metabolic related processes. Through further target validation and biological function studies, this study hopes to systemically investigate of HNF4α’s role in GC tumorigenesis, especially in metabolism related pathways. Citation Format: Chang Xu, Wen Fong Ooi, Aditi Qamra, Patrick Tan. Functional characterization of HNF4α in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3559. doi:10.1158/1538-7445.AM2017-3559

Research progress of radiotherapy and molecular classifications of gastric cancer

Adjuvant radiotherapy (RT) can improve the rate of loco-regional control for patients with gastric cancer (GC), while the selection of patients plays a key role. As the research moves along, several relatively comprehensive molecular classifications emerged such as the TCGA classification and the ACRG classification. Studies have demonstrated that molecular classifications are closely related to the clinicopathologic characteristics, prognosis and treatment response. However, there is not recognized molecular classification of GC presently. It is a great challenge for radiation oncologists to make use of the individual bioinformation and accurately select patients who would benefit from RT. Meanwhile, precision RT could also be achieved with the prediction of radiosensitivity, combination of RT with targeted therapy and the application of ctDNA within the field of RT. Key words: Stomach neoplasms/RT/PA; DNA, neoplasm; Review

The HDAC Inhibitors Scriptaid and LBH589 Combined with the Oncolytic Virus Delta24-RGD Exert Enhanced Anti-Tumor Efficacy in Patient-Derived Glioblastoma Cells.

BackgroundA phase I/II trial for glioblastoma with the oncolytic adenovirus Delta24-RGD was recently completed. Delta24-RGD conditionally replicates in cells with a disrupted retinoblastoma-pathway and enters cells via αvβ3/5 integrins. Glioblastomas are differentially sensitive to Delta24-RGD. HDAC inhibitors (HDACi) affect integrins and share common cell death pathways with Delta24-RGD. We studied the combination treatment effects of HDACi and Delta24-RGD in patient-derived glioblastoma stem-like cells (GSC), and we determined the most effective HDACi.MethodsSAHA, Valproic Acid, Scriptaid, MS275 and LBH589 were combined with Delta24-RGD in fourteen distinct GSCs. Synergy was determined by Chou Talalay method. Viral infection and replication were assessed using luciferase and GFP encoding vectors and hexon-titration assays. Coxsackie adenovirus receptor and αvβ3 integrin levels were determined by flow cytometry. Oncolysis and mechanisms of cell death were studied by viability, caspase-3/7, LDH and LC3B/p62, phospho-p70S6K. Toxicity was studied on normal human astrocytes. MGMT promotor methylation status, TCGA classification, Rb-pathway and integrin gene expression levels were assessed as markers of responsiveness.ResultsScriptaid and LBH589 acted synergistically with Delta24-RGD in approximately 50% of the GSCs. Both drugs moderately increased αvβ3 integrin levels and viral infection in responding but not in non-responding GSCs. LBH589 moderately increased late viral gene expression, however, virus titration revealed diminished viral progeny production by both HDACi, Scriptaid augmented caspase-3/7 activity, LC3B conversion, p62 and phospho-p70S6K consumption, as well as LDH levels. LBH589 increased LDH and phospho-p70S6K consumption. Responsiveness correlated with expression of various Rb-pathway genes and integrins. Combination treatments induced limited toxicity to human astrocytes.ConclusionLBH589 and Scriptaid combined with Delta24-RGD revealed synergistic anti-tumor activity in a subset of GSCs. Both HDACi moderately augmented viral infection and late gene expression, but slightly reduced progeny production. The drugs differentially activated multiple cell death pathways. The limited toxicity on astrocytes supports further evaluation of the proposed combination therapies.

Prognostic and therapeutic relevance of molecular subtypes in high-grade serous ovarian cancer.

Molecular classification of high-grade serous ovarian cancer (HGSOC) using transcriptional profiling has proven to be complex and difficult to validate across studies. We determined gene expression profiles of 174 well-annotated HGSOCs and demonstrate prognostic significance of the prespecified TCGA Network gene signatures. Furthermore, we confirm the presence of four HGSOC transcriptional subtypes using a de novo classification. Survival differed statistically significantly between de novo subtypes (log rank, P = .006) and was the best for the immunoreactive-like subtype, but statistically significantly worse for the proliferative- or mesenchymal-like subtypes (adjusted hazard ratio = 1.89, 95% confidence interval = 1.18 to 3.02, P = .008, and adjusted hazard ratio = 2.45, 95% confidence interval = 1.43 to 4.18, P = .001, respectively). More prognostic information was provided by the de novo than the TCGA classification (Likelihood Ratio tests, P = .003 and P = .04, respectively). All statistical tests were two-sided. These findings were replicated in an external data set of 185 HGSOCs and confirm the presence of four prognostically relevant molecular subtypes that have the potential to guide therapy decisions.

Prognostic relevance of gene signatures in high-grade serous ovarian carcinoma.

5510 Background: Transcriptional profiling of ovarian cancers has proven to be complex. As such it has been difficult to validate existing signatures across studies. Cancer Genome Atlas (TCGA) researchers have identified four molecular subtypes of high-grade serous ovarian cancer (HGSOC). However, survival duration did not differ significantly for the TCGA subtypes. Potential limitations of the TCGA data include short clinical follow-up (45% were alive at the time of last follow-up) and the need to unify gene expression measures from multiple platforms. Methods: Clinically annotated stage-II–IV HGSOC samples (n = 175) with >70% tumor cell content were profiled using the Agilent Whole Human Genome 4 x 44K chip. To identify subtypes non-negative matrix factorization (NMF) of mRNA expression was performed using ~2000 genes with the highest variability across patients. In parallel differentially expressed genes were identified using the Rosetta Similarity Search Tool as well as analysis of variance based on genes known to be involved in epithelial to mesenchymal transition. Results: Median follow-up time was 35 months (range, 0–202 months, 12% were alive at the time of last follow-up). NMF clustering confirmed four HGSOC subtypes (immunoreactive, differentiated, proliferative, and mesenchymal) on the basis of gene content in the clusters. Pathway signatures with therapeutic potential were identified for individual or multiple subtypes. Survival differed significantly between the four molecular subgroups in univariate (Hazard Ratio [HR] 2.4, 95% CI 1.5-4.1, p = 0.007) and multivariate (HR 2.3, 95% CI 1.3-4.0, p = 0.003) analyses when accounting for age, stage, grade, and postoperative residual tumor. Using the supervised clustering approach two distinct molecular subtypes of HGSOC based on epithelial and mesenchymal gene expression signatures were identified with significantly different survival outcomes. Conclusions: Here we independently validate and expand upon the molecular TCGA classification of HGSOC. The potential of these two prognostic classifiers may lie in their ability to recognize categories of patients that are more likely to respond to particular therapies.