Yujin Hoshida

Gene expression in fixed tissues and outcome in hepatocellular carcinoma.

BACKGROUND It is a challenge to identify patients who, after undergoing potentially curative treatment for hepatocellular carcinoma, are at greatest risk for recurrence. Such high-risk patients could receive novel interventional measures. An obstacle to the development of genome-based predictors of outcome in patients with hepatocellular carcinoma has been the lack of a means to carry out genomewide expression profiling of fixed, as opposed to frozen, tissue. METHODS We aimed to demonstrate the feasibility of gene-expression profiling of more than 6000 human genes in formalin-fixed, paraffin-embedded tissues. We applied the method to tissues from 307 patients with hepatocellular carcinoma, from four series of patients, to discover and validate a gene-expression signature associated with survival. RESULTS The expression-profiling method for formalin-fixed, paraffin-embedded tissue was highly effective: samples from 90% of the patients yielded data of high quality, including samples that had been archived for more than 24 years. Gene-expression profiles of tumor tissue failed to yield a significant association with survival. In contrast, profiles of the surrounding nontumoral liver tissue were highly correlated with survival in a training set of tissue samples from 82 Japanese patients, and the signature was validated in tissues from an independent group of 225 patients from the United States and Europe (P=0.04). CONCLUSIONS We have demonstrated the feasibility of genomewide expression profiling of formalin-fixed, paraffin-embedded tissues and have shown that a reproducible gene-expression signature correlated with survival is present in liver tissue adjacent to the tumor in patients with hepatocellular carcinoma.

Lin28 promotes transformation and is associated with advanced human malignancies

Multiple members of the let-7 family of miRNAs are often repressed in human cancers, thereby promoting oncogenesis by derepressing targets such as HMGA2, K-Ras and c-Myc. However, the mechanism by which let-7 miRNAs are coordinately repressed is unclear. The RNA-binding proteins LIN28 and LIN28B block let-7 precursors from being processed to mature miRNAs, suggesting that their overexpression might promote malignancy through repression of let-7. Here we show that LIN28 and LIN28B are overexpressed in primary human tumors and human cancer cell lines (overall frequency ∼15%), and that overexpression is linked to repression of let-7 family miRNAs and derepression of let-7 targets. LIN28 and LIN28b facilitate cellular transformation in vitro, and overexpression is associated with advanced disease across multiple tumor types. Our work provides a mechanism for the coordinate repression of let-7 miRNAs observed in a subset of human cancers, and associates activation of LIN28 and LIN28B with poor clinical prognosis.

TMPRSS2:ERG gene fusion associated with lethal prostate cancer in a watchful waiting cohort.

The identification of the TMPRSS2:ERG fusion in prostate cancer suggests that distinct molecular subtypes may define risk for disease progression. In surgical series, TMPRSS2:ERG fusion was identified in 50% of the tumors. Here, we report on a population-based cohort of men with localized prostate cancers followed by expectant (watchful waiting) therapy with 15% (17/111) TMPRSS2:ERG fusion. We identified a statistically significant association between TMPRSS2:ERG fusion and prostate cancer specific death (cumulative incidence ratio=2.7, P<0.01, 95% confidence interval=1.3–5.8). Quantitative reverse-transcription–polymerase chain reaction demonstrated high estrogen-regulated gene (ERG) expression to be associated with TMPRSS2:ERG fusion (P<0.005). These data suggest that TMPRSS2:ERG fusion prostate cancers may have a more aggressive phenotype, possibly mediated through increased ERG expression.

Pivotal Role of mTOR Signaling in Hepatocellular Carcinoma

BACKGROUND & AIMS The advent of targeted therapies in hepatocellular carcinoma (HCC) has underscored the importance of pathway characterization to identify novel molecular targets for treatment. We evaluated mTOR signaling in human HCC, as well as the antitumoral effect of a dual-level blockade of the mTOR pathway. METHODS The mTOR pathway was assessed using integrated data from mutation analysis (direct sequencing), DNA copy number changes (SNP-array), messenger RNA levels (quantitative reverse-transcription polymerase chain reaction and gene expression microarray), and protein activation (immunostaining) in 351 human samples [HCC (n = 314) and nontumoral tissue (n = 37)]. Effects of dual blockade of mTOR signaling using a rapamycin analogue (everolimus) and an epidermal/vascular endothelial growth factor receptor inhibitor (AEE788) were evaluated in liver cancer cell lines and in a xenograft model. RESULTS Aberrant mTOR signaling (p-RPS6) was present in half of the cases, associated with insulin-like growth factor pathway activation, epidermal growth factor up-regulation, and PTEN dysregulation. PTEN and PI3KCA-B mutations were rare events. Chromosomal gains in RICTOR (25% of patients) and positive p-RPS6 staining correlated with recurrence. RICTOR-specific siRNA down-regulation reduced tumor cell viability in vitro. Blockage of mTOR signaling with everolimus in vitro and in a xenograft model decelerated tumor growth and increased survival. This effect was enhanced in vivo after epidermal growth factor blockade. CONCLUSIONS MTOR signaling has a critical role in the pathogenesis of HCC, with evidence for the role of RICTOR in hepato-oncogenesis. MTOR blockade with everolimus is effective in vivo. These findings establish a rationale for targeting the mTOR pathway in clinical trials in HCC.

Integrative Transcriptome Analysis Reveals Common Molecular Subclasses of Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a highly heterogeneous disease, and prior attempts to develop genomic-based classification for HCC have yielded highly divergent results, indicating difficulty in identifying unified molecular anatomy. We performed a meta-analysis of gene expression profiles in data sets from eight independent patient cohorts across the world. In addition, aiming to establish the real world applicability of a classification system, we profiled 118 formalin-fixed, paraffin-embedded tissues from an additional patient cohort. A total of 603 patients were analyzed, representing the major etiologies of HCC (hepatitis B and C) collected from Western and Eastern countries. We observed three robust HCC subclasses (termed S1, S2, and S3), each correlated with clinical parameters such as tumor size, extent of cellular differentiation, and serum alpha-fetoprotein levels. An analysis of the components of the signatures indicated that S1 reflected aberrant activation of the WNT signaling pathway, S2 was characterized by proliferation as well as MYC and AKT activation, and S3 was associated with hepatocyte differentiation. Functional studies indicated that the WNT pathway activation signature characteristic of S1 tumors was not simply the result of beta-catenin mutation but rather was the result of transforming growth factor-beta activation, thus representing a new mechanism of WNT pathway activation in HCC. These experiments establish the first consensus classification framework for HCC based on gene expression profiles and highlight the power of integrating multiple data sets to define a robust molecular taxonomy of the disease.

SMAD4-dependent barrier constrains prostate cancer growth and metastatic progression

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP–SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal versus distal colorectum

Objective Colorectal cancer is typically classified into proximal colon, distal colon and rectal cancer. Tumour genetic and epigenetic features differ by tumour location. Considering a possible role of bowel contents (including microbiome) in carcinogenesis, this study hypothesised that tumour molecular features might gradually change along bowel subsites, rather than change abruptly at splenic flexure. Design Utilising 1443 colorectal cancers in two US nationwide prospective cohort studies, the frequencies of molecular features (CpG island methylator phenotype (CIMP), microsatellite instability (MSI), LINE-1 methylation and BRAF, KRAS and PIK3CA mutations) were examined along bowel subsites (rectum, rectosigmoid junction, sigmoid, descending colon, splenic flexure, transverse colon, hepatic flexure, ascending colon and caecum). The linearity and non-linearity of molecular relations along subsites were statistically tested by multivariate logistic or linear regression analysis. Results The frequencies of CIMP-high, MSI-high and BRAF mutations gradually increased from the rectum (<2.3%) to ascending colon (36–40%), followed by falls in the caecum (12–22%). By linearity tests, these molecular relations were significantly linear from rectum to ascending colon (p<0.0001), and there was little evidence of non-linearity (p>0.09). Caecal cancers exhibited the highest frequency of KRAS mutations (52% vs 27–35% in other sites; p<0.0001). Conclusions The frequencies of CIMP-high, MSI-high and BRAF mutations in cancer increased gradually along colorectum subsites from the rectum to ascending colon. These novel data challenge the common conception of discrete molecular features of proximal versus distal colorectal cancers, and have a substantial impact on clinical, translational and epidemiology research, which has typically been performed with the dichotomous classification of proximal versus distal tumours.

Genomic sequencing of colorectal adenocarcinomas identifies a recurrent VTI1A-TCF7L2 fusion

Prior studies have identified recurrent oncogenic mutations in colorectal adenocarcinoma and have surveyed exons of protein-coding genes for mutations in 11 affected individuals. Here we report whole-genome sequencing from nine individuals with colorectal cancer, including primary colorectal tumors and matched adjacent non-tumor tissues, at an average of 30.7× and 31.9× coverage, respectively. We identify an average of 75 somatic rearrangements per tumor, including complex networks of translocations between pairs of chromosomes. Eleven rearrangements encode predicted in-frame fusion proteins, including a fusion of VTI1A and TCF7L2 found in 3 out of 97 colorectal cancers. Although TCF7L2 encodes TCF4, which cooperates with β-catenin in colorectal carcinogenesis, the fusion lacks the TCF4 β-catenin–binding domain. We found a colorectal carcinoma cell line harboring the fusion gene to be dependent on VTI1A-TCF7L2 for anchorage-independent growth using RNA interference-mediated knockdown. This study shows previously unidentified levels of genomic rearrangements in colorectal carcinoma that can lead to essential gene fusions and other oncogenic events.

Molecular classification and novel targets in hepatocellular carcinoma: recent advancements.

Hepatocellular carcinoma (HCC) is one of most lethal cancers worldwide. Strategic decisions for the advancement of molecular therapies in this neoplasm require a clear understanding of its molecular classification. Studies indicate aberrant activation of signaling pathways involved in cellular proliferation (e.g., epidermal growth factor and RAS/mitogen-activated protein kinase pathways), survival (e.g., Akt/mechanistic target of rapamycin pathway), differentiation (e.g., Wnt and Hedgehog pathways), and angiogenesis (e.g., vascular endothelial growth factor and platelet-derived growth factor), which is heterogeneously presented in each tumor. Integrative analysis of accumulated genomic datasets has revealed a global scheme of molecular classification of HCC tumors observed across diverse etiologic factors and geographic locations. Such a framework will allow systematic understanding of the frequently co-occurring molecular aberrations to design treatment strategy for each specific subclass of tumors. Accompanied by a growing number of clinical trials of molecular targeted drugs, diagnostic and prognostic biomarker development will be facilitated with special attention on study design and with new assay technologies specialized for archived fixed tissues. A new class of genomic information, microRNA dysregulation and epigenetic alterations, will provide insight for more precise understanding of disease mechanism and expand the opportunity of biomarker/therapeutic target discovery. These efforts will eventually enable personalized management of HCC.

Combining Clinical, Pathology, and Gene Expression Data to Predict Recurrence of Hepatocellular Carcinoma

BACKGROUND & AIMS In approximately 70% of patients with hepatocellular carcinoma (HCC) treated by resection or ablation, disease recurs within 5 years. Although gene expression signatures have been associated with outcome, there is no method to predict recurrence based on combined clinical, pathology, and genomic data (from tumor and cirrhotic tissue). We evaluated gene expression signatures associated with outcome in a large cohort of patients with early stage (Barcelona-Clinic Liver Cancer 0/A), single-nodule HCC and heterogeneity of signatures within tumor tissues. METHODS We assessed 287 HCC patients undergoing resection and tested genome-wide expression platforms using tumor (n = 287) and adjacent nontumor, cirrhotic tissue (n = 226). We evaluated gene expression signatures with reported prognostic ability generated from tumor or cirrhotic tissue in 18 and 4 reports, respectively. In 15 additional patients, we profiled samples from the center and periphery of the tumor, to determine stability of signatures. Data analysis included Cox modeling and random survival forests to identify independent predictors of tumor recurrence. RESULTS Gene expression signatures that were associated with aggressive HCC were clustered, as well as those associated with tumors of progenitor cell origin and those from nontumor, adjacent, cirrhotic tissues. On multivariate analysis, the tumor-associated signature G3-proliferation (hazard ratio [HR], 1.75; P = .003) and an adjacent poor-survival signature (HR, 1.74; P = .004) were independent predictors of HCC recurrence, along with satellites (HR, 1.66; P = .04). Samples from different sites in the same tumor nodule were reproducibly classified. CONCLUSIONS We developed a composite prognostic model for HCC recurrence, based on gene expression patterns in tumor and adjacent tissues. These signatures predict early and overall recurrence in patients with HCC, and complement findings from clinical and pathology analyses.