Christopher J. Lafargue

SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas

Lineage-survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development. Here we show that a peak of genomic amplification on chromosome 3q26.33 found in squamous cell carcinomas (SCCs) of the lung and esophagus contains the transcription factor gene SOX2, which is mutated in hereditary human esophageal malformations, is necessary for normal esophageal squamous development, promotes differentiation and proliferation of basal tracheal cells and cooperates in induction of pluripotent stem cells. SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 here cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These characteristics identify SOX2 as a lineage-survival oncogene in lung and esophageal SCC.

Oncosome Formation in Prostate Cancer: Association with a Region of Frequent Chromosomal Deletion in Metastatic Disease

Oncosomes have recently been described as membrane-derived microvesicles secreted by cancer cells, which transfer oncogenic signals and protein complexes across cell boundaries. Here, we show the rapid formation and secretion of oncosomes from DU145 and LNCaP human prostate cancer cells. Oncosome formation was stimulated by epidermal growth factor receptor activation and also by overexpression of membrane-targeted Akt1. Microvesicles shed from prostate cancer cells contained numerous signal transduction proteins and were capable of activating rapid phospho-tyrosine and Akt pathway signaling, and stimulating proliferation and migration, in recipient tumor cells. They also induced a stromal reaction in recipient normal cells. Knockdown of the actin nucleating protein Diaphanous Related Formin 3 (DRF3/Dia2) by RNA interference enhanced rates of oncosome formation, indicating that these structures resemble, and may be identical to, nonapoptotic membrane blebs, a feature of the amoeboid form of cell motility. Analysis of primary and metastatic human prostate tumors using 100K single nucleotide polymorphism arrays revealed a significantly higher frequency of deletion of the locus encoding DRF3 (DIAPH3) in metastatic tumors (P = 0.001) in comparison with organ-confined tumors. Fluorescence in situ hybridization confirmed increased chromosomal loss of DIAPH3 in metastatic tumors in a different cohort of patients (P = 0.006). These data suggest that microvesicles shed from prostate cancer cells can alter the tumor microenvironment in a manner that may promote disease progression. They also show that DRF3 is a physiologically relevant protein that seems to regulate this process.

Prevalence of TMPRSS2-ERG and SLC45A3-ERG gene fusions in a large prostatectomy cohort

The majority of prostate cancers harbor recurrent gene fusions between the hormone-regulated TMPRSS2 and members of the ETS family of transcription factors, most commonly ERG. Prostate cancer with ERG rearrangements represent a distinct sub-class of tumor based on studies reporting associations with histomorphologic features, characteristic somatic copy number alterations, and gene expression signatures. This study describes the frequency of ERG rearrangement prostate cancer and three 5 prime (5′) gene fusion partners (ie, TMPRSS2, SLC45A3, and NDRG1) in a large prostatectomy cohort. ERG gene rearrangements and mechanism of rearrangement, as well as rearrangements of TMPRSS2, SLC45A3, and NDRG1, were assessed using fluorescence in situ hybridization (FISH) on prostate cancer samples from 614 patients treated using radical prostatectomy. ERG rearrangement occurred in 53% of the 540 assessable cases. TMPRSS2 and SLC45A3 were the only 5′ partner in 78% and 6% of these ERG rearranged cases, respectively. Interestingly, 11% of the ERG rearranged cases showed concurrent TMPRSS2 and SLC45A3 rearrangements. TMPRSS2 or SLC45A3 rearrangements could not be identified for 5% of the ERG rearranged cases. From these remaining cases we identified one case with NDRG1 rearrangement. We did not observe any associations with pathologic parameters or clinical outcome. This is the first study to describe the frequency of SLC45A3–ERG fusions in a large clinical cohort. Most studies have assumed that all ERG rearranged prostate cancers harbor TMPRSS2–ERG fusions. This is also the first study to report concurrent TMPRSS2 and SLC45A3 rearrangements in the same tumor focus, suggesting additional complexity that had not been previously appreciated. This study has important clinical implications for the development of diagnostic assays to detect ETS rearranged prostate cancer. Incorporation of these less common ERG rearranged prostate cancer fusion assays could further increase the sensitivity of the current PCR-based approaches.

Distinct Genomic Aberrations Associated With ERG Rearranged Prostate Cancer

Emerging molecular and clinical data suggest that ETS fusion prostate cancer represents a distinct molecular subclass, driven most commonly by a hormonally regulated promoter and characterized by an aggressive natural history. The study of the genomic landscape of prostate cancer in the light of ETS fusion events is required to understand the foundation of this molecularly and clinically distinct subtype. We performed genome‐wide profiling of 49 primary prostate cancers and identified 20 recurrent chromosomal copy number aberrations, mainly occurring as genomic losses. Co‐occurring events included losses at 19q13.32 and 1p22.1. We discovered three genomic events associated with ERG rearranged prostate cancer, affecting 6q, 7q, and 16q. 6q loss in nonrearranged prostate cancer is accompanied by gene expression deregulation in an independent dataset and by protein deregulation of MYO6. To analyze copy number alterations within the ETS genes, we performed a comprehensive analysis of all 27 ETS genes and of the 3 Mbp genomic area between ERG and TMPRSS2 (21q) with an unprecedented resolution (30 bp). We demonstrate that high‐resolution tiling arrays can be used to pin‐point breakpoints leading to fusion events. This study provides further support to define a distinct molecular subtype of prostate cancer based on the presence of ETS gene rearrangements.

TTF1 expression in non‐small cell lung carcinoma: association with TTF1 gene amplification and improved survival

Acquired chromosomal aberrations play an important role in tumour development and progression. Such genetic alterations occur in a significant proportion of non‐small cell lung carcinomas (NSCLCs) and include amplification of 14q13.3, which contains the TTF1 gene. We asked whether TTF1 amplification is associated with increased TTF1 protein expression in NSCLCs, and whether TTF1 is associated with clinicopathological features, including patient survival. We used a FISH assay and quantitative immunohistochemical staining to interrogate a population‐based cohort of 538 NSCLCs from Swiss patients for TTF1 amplification and protein expression. We found TTF1 amplification in ∼13% of adenocarcinomas (ACs) and in ∼9% of squamous cell carcinomas (SCCs) and TTF1 amplification was associated with increased TTF1 protein expression. High‐level TTF1 expression was significantly associated with smaller tumour size, female gender and longer overall survival only among ACs (median survival 82 versus 28 months; p = 0.002). On multivariate analysis, high TTF1 expression was an independent predictor of favourable prognosis in patients with AC [hazard ratio, 0.56 (95% CI 0.38–0.83); p = 0.008]. We conclude that TTF1 amplification is a mechanism of high‐level TTF1 expression in a subset of NSCLCs. When expressed at high levels, this routinely used diagnostic marker is also an independent biomarker of favourable prognosis in AC. Copyright

Amplification of chromosomal segment 4q12 in non-small cell lung cancer

In cancer, proto-oncogenes are often altered by genomic amplification. Here we report recurrent focal amplifications of chromosomal segment 4q12 overlapping the proto-oncogenes PDGFRA and KIT in non-small cell lung cancer (NSCLC). Single nucleotide polymorphism (SNP) array and fluorescent in situ hybridization (FISH) analysis indicate that 4q12 is amplified in 3-7% of lung adenocarcinomas and 8-10% of lung squamous cell carcinomas. In addition, we demonstrate that the NSCLC cell line NCI-H1703 exhibits focal amplification of PDGFRA and is dependent on PDGFRα activity for cell growth. Treatment of NCI-H1703 cells with PDGFRA-specific shRNAs or with the PDGFRα/KIT small molecule inhibitors imatinib or sunitinib leads to cell growth inhibition. However, these observations do not extend to NSCLC cell lines with lower-amplitude and broader gains of chromosome 4q. Together these observations implicate PDGFRA and KIT as potential oncogenes in NSCLC, but further study is needed to define the specific characteristics of those tumors that could respond to PDGFRα/KIT inhibitors.

Testing mutual exclusivity of ETS rearranged prostate cancer

Prostate cancer is a clinically heterogeneous and multifocal disease. More than 80% of patients with prostate cancer harbor multiple geographically discrete cancer foci at the time of diagnosis. Emerging data suggest that these foci are molecularly distinct consistent with the hypothesis that they arise as independent clones. One of the strongest arguments is the heterogeneity observed in the status of E26 transformation specific (ETS) rearrangements between discrete tumor foci. The clonal evolution of individual prostate cancer foci based on recent studies demonstrates intertumoral heterogeneity with intratumoral homogeneity. The issue of multifocality and interfocal heterogeneity is important and has not been fully elucidated due to lack of the systematic evaluation of ETS rearrangements in multiple tumor sites. The current study investigates the frequency of multiple gene rearrangements within the same focus and between different cancer foci. Fluorescence in situ hybridization (FISH) assays were designed to detect the four most common recurrent ETS gene rearrangements. In a cohort of 88 men with localized prostate cancer, we found ERG, ETV1, and ETV5 rearrangements in 51% (44/86), 6% (5/85), and 1% (1/86), respectively. None of the cases demonstrated ETV4 rearrangements. Mutual exclusiveness of ETS rearrangements was observed in the majority of cases; however, in six cases, we discovered multiple ETS or 5′ fusion partner rearrangements within the same tumor focus. In conclusion, we provide further evidence for prostate cancer tumor heterogeneity with the identification of multiple concurrent gene rearrangements.

TMPRSS2-ERG fusion is frequently observed in Gleason pattern 3 prostate cancer in a Canadian cohort.

Purpose: TMPRSS2-ERG gene fusion was recently reported as the most common gene rearrangement in prostate cancer (PCA). Experimental Design: Using break-apart FISH assay to indirectly assess the fusion of TMPRSS2-ERG. We sought to characterize the incidence, pathological features and clinical parameters of TMPRSS2-ERG gene fusion in a cohort of 196 Canadian men treated by radical prostatectomy for localized PCA, and to investigate its potential as a biomarker in PCA. Results: In this cohort 41% of the patients showed positive gene fusion status in their PCA. The TMPRSS2-ERG gene fusion status was homogenous within the same cancer focus and 82% of fusion positive PCA were present in GS 6 or 7 vs. 14% in GS 8 (p= 0.004). Moreover, TMPRSS2-ERG fusion was present in 42% of Gleason pattern 3 vs. 27% of Gleason pattern 4 (p = 0.014). However, in this study, no significant association was noticed between TMPRSS2-ERG fusion status in relation to pathological stage, surgical margin or biochemical failure. Conclusion: The higher association of TMPRSS2-ERG with Gleason score 6 and 7 should be further investigated. If confirmed, this could have significant clinical impact in further stratifying patients with PCA should the TMPRSS2-ERG be confirmed as a prognostic biomarker. >p/>

Abstract LB-21: Emergence of castration resistant prostate cancer class defined by recurrentERGfusion

The role of recurrent ETS gene fusions in castration resistant prostate cancer (CRPC) is poorly understood. We characterized the transcriptome of CRPC by interrogating a cohort of 54 men with locally advanced or metastatic CRPC using a gene expression profiling method recently established for formalin-fixed paraffin-embedded tissue. We found increased expression of multiple genes in ERG rearranged tumors which suggests a selective growth advantage under oxygen poor conditions. We discovered that trefoil factor 3 (TFF3) is the top differentially regulated gene by both ERG rearrangement and resistance to therapy. Conventional chromatin immunoprecipitation (ChIP)-PCR and ChIP-seq data revealed direct binding of ERG to ETS binding sites in the TFF3 promoter in ERG rearranged prostate cancer cell lines, which was confirmed in ERG rearranged hormone naive prostate cancer (HNPC) and CRPC tissue samples. In vitro data suggested that ERG has an inhibitory effect on TFF3 expression in hormone naive cancer, which was relieved in CRPC depending on the level of AR expression. These findings reveal an interplay between ERG rearrangement and AR signaling in CRPC, and provide perspective towards novel therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-21.